The Team Aqua/Magma Leader Battle theme music featured in Pokémon Ruby, Sapphire and Emerald is based on the Shionchou boss battle theme music from Game Freak's Super Famicom game Bushi Seiryūden: Futari no Yūsha. The themes were written by both games' composer and Game Freak co-founder Junichi Masuda.

When traveling on the cable car, there is a 1 in 64 chance for an NPC to appear in the background - a hiker, a camper, or a picnicker. However, there also exists a 4th NPC which never appears due to a faulty line of code. These include a Zigzagoon in Emerald, and a Poochyena in Ruby and Sapphire

The opening battle against the Poochyena attacking Professor Birch and the player's first Pokémon cannot be lost and the Poochyena cannot win. The Poochyena is programmed to flee the battle if the player somehow manages to get their Starter to red health.

The games' most prominent subplot focuses on the conflict between Team Aqua and Team Magma in their efforts to gain control of the legendary Pokémon Kyogre and Groudon. Their goals are to use them to change the climate in response to the effect of humans on the environment in order to create expanded environments for sea and land Pokémon respectively. Although it has not been confirmed by the developers, this subplot may have drawn direct inspiration from a real-life controversy that continues to be a prominent issue in Japan. The Isahaya Bay land reclamation project on the island of Kyūshū, which the Hoenn region is based on, aimed to expand the available farmland in one of Japan's last wetland habitats. This led to fierce political conflict from environmentalists who argued that the project would cause long-term damage to the wetlands and the marine ecosystem of the area through agricultural runoff released into the sea, and from reclamation activists who argued that Kyūshū needed the land as Japan has very little arable land already and needs to produce enough food to feed its increasing population and keep up with rapid industrialization. The concept of Team Aqua and Team Magma draw general similarities to each side of this issue (i.e. reclaiming land where there used to be sea and vice versa) while being written as cultic villains akin to Team Rocket from past games without distinct arguments to their positions. This causes these similarities to be obscured and emphasizes the personal gain of expanding, or reducing land for the sake of certain land, or sea Pokémon to be won out from the conflict with little to no regard for humanity.

In Pokémon Emerald, the unified story featuring Kyogre and Groudon both being pacified by the presence of Rayquaza, a Pokémon heralding from the sky which in many religions and mythologies is where powerful gods and deities live, hints that a divine compromise between civilization and nature is the necessary solution. This compromise is seen with how Hoenn is presented in the final game, with the coexistence of different environments, humans and Pokémon being the result. This suggests that the preservation of Isahaya Bay while allowing for land reclamation elsewhere is the compromise this subplot is trying to get across.

In May 2020, a Japanese livestreamer began a lengthy series using his two pet Siamese fighting fish to play through Pokemon Ruby & Sapphire using a system involving several programs connected to the game's emulator. These programs rely on a webcam to track the fish's movements and determine what actions to perform by where it swims over a map placed behind the fish tank, so wherever the fish swims, it blocks a map square with a picture of a controller input on it and the system performs said input in-game.

On October 3, 2020, during a stream playing through Pokemon Sapphire, one of the fish named Mutekimaru was working on a boulder puzzle in the Seafloor Cavern on Route 128 when it performed a glitch that appeared to have not been widely known in the past. The fish used Strength on a boulder, which moved it and additionally created a duplicate boulder in its place, soft-locking the puzzle until the room is reset. The streamer later figured out how to trigger the glitch himself, and uploaded a step-by-step guide to YouTube on how to perform it.

Kiri, the girl in Sootopolis City who gives the player two berries each day, was named after director Junichi Masuda's daughter. Kiri was born in September 2002, just two months before the Japanese release of Pokemon Ruby & Sapphire.

Ruby & Sapphire feature various unused music tracks from towns in Gold, Silver and Crystal, along with multiple incomplete versions of the contest music.

Some of the trainer sprites in Ruby, Sapphire, and Emerald were changed for the international versions. Hex Maniacs were given smaller eyes with pupils, and the female Psychics have their arms outstretched, and the Male Cooltrainer has minor changes to his hairstyle.
The most obvious change is the sailor, whose original pose resembles a gesture called the "Bras d'honneur", involving raising a fist and slapping the biceps on the same arm (also known as the "Iberian slap" or "Iberian finger"), which is seen as an offensive gesture in a number of countries.

If the player lacks the 50 Pokédollar fee to enter the Oceanic Museum while Team Aqua or Team Magma occupies it, they will be admitted for free, as the receptionist will mistake the player for a member of Team Aqua/Magma. This will no longer work after Team Aqua/Magma leaves the building, however.

Two old women could be found in the hot spring of Lavaridge Town in the original release of Pokémon Ruby & Pokémon Sapphire. They were removed in the Nintendo 3DS remakes, possibly because of the lewd nature of the scene. In Japan, it is common for people to bathe naked in hot springs, called "onsen", split by gender. In the original games' release, the male trainer was able to bathe with the old women, while an old man is also seen looking over the spring.

Wild Double Battles were planned for Ruby and Sapphire as evidenced by an unused text string found in the game. The string reads, "Wild v[&H05] and v[&H03] appeared!", with v[&H05] and v[&H03] being placeholders for the names of the Pokemon.

In Victory Road, there is a one-way ledge on B1F. However, in the Spanish, Italian, French and German releases the top tile of the ledge was removed to allow access either way, and in Emerald, the entire ledge was removed.

The gems on Sableye's body may be a reference to Pokemon Ruby and Sapphire, as the common colors for rubies and sapphires are red and blue. Sableye's shiny sprite also has green and yellow gems. Green is the common color of emeralds, making this possible reference also line up with Pokemon Emerald.

This is the only main series game where the player character's biological father is shown. In Ruby & Sapphire, the gym leader Norman is the player character's father, regardless of which gender is selected.

Examining the TV in the player's house describes a movie with "Two men dancing on a giant piano keyboard." This most likely refers to a scene from the movie "Big".

These were the first Pokémon games to have a framerate of 60 frames per second (FPS). This change carried over to FireRed, LeafGreen, and Emerald. However, the framerate was downgraded to 30 FPS in the Generation IV games

An early version of the Ruby & Sapphire box art shows the version name under the cover Pokemon and the inclusion of the "Gotta catch em all!" slogan. In the final version, the version name was moved to the top and the slogan was removed entirely.

Nincada is the only Pokemon who will evolve into two different Pokemon at once. When Nincada evolves, if there is an empty slot in the player's party and a pokeball in their inventory, the player will receive both Ninjask and Shedinja. additionally, if Nincada is shiny, Ninjask and Shedinja will also be shiny.

Gastrodon and Shellos were supposed to appear in Pokemon Ruby & Sapphire. Due to time constraints they weren't added. Ken Sugimori only did one version of the Pokemon (there are two varieties in Diamond and Pearl).

In Ruby, Sapphire, and Emerald, there is a man in the Devon Corp. that speaks about entering or seeing Pokemon dreams. This becomes possible with the introduction of the Dream World in generation V.