In the SNES game, Mario and Luigi can not spin jump on some enemies that they can in the Game Boy Advance game, such as Boos and Podoboos. Also, in the Game Boy Advance game, Yoshi can jump off of fish enemies.

By holding the run button, Mario and Luigi can climb vines faster in the Game Boy Advance game.

In the SNES game, Yoshi will always produce an egg containing a mushroom when he eats ten red berries. In the Game Boy Advance version Green Yoshi will produce an egg with a mushroom, Blue Yoshi will produce an egg with a feather, Red Yoshi will produce an egg with a Fire Flower, and Yellow Yoshi will produce and egg with a Starman.

In the SNES game, Yoshi has orange arms regardless of his body color, though he has green arms on the SNES box art. In the Game Boy Advance version, Yoshi's arms are the same color as his body.

In the SNES game, player 1 controls Mario and player 2 controls Luigi. In the Game Boy Advance game, the player can choose to play as either Mario or Luigi by pressing the R button on the world map. Additionally, in the SNES version Luigi is an exact copy of Mario with a different color palette. In the Game Boy Advance version, Luigi was changed in several ways including:
• Luigi is taller and skinnier than Mario and more closely resembles his modern apperance. Luigi also wears blue overalls instead of his purple overalls from the SNES version.
• Luigi jumps higher, moves slower, and scuttles in mid-air much like he does in Super Mario Bros. 2 (U.S. version). He also runs slower and has worse traction than Mario.
• Luigi flies higher with his cape than Mario, but also flies slower.
• Fireballs from Fire Luigi bounce higher.
• When Luigi rides Yoshi, Yoshi doesn't instantly eat enemies; Yoshi can spit enemies out, and use some of them as weapons, but swallows them after 7-8 seconds.
• All coins will fall out of a block if Luigi hits it, whereas the coins come out one-by-one when Mario hits the same block.

In the Game Boy Advance game, earning at least 10 lives consecutively will cause a flower and the number of lives earned to scroll across the screen.

In the SNES game, the player can only save after completing certain stages or finishing any Ghost House or Fortress. In the Game Boy Advance game, the player can save inside of a course or on the world map; the Game Boy Advance game also saves the player's power-ups and life count, whereas the SNES version starts the player out as Small Mario with 5 lives each time a saved file is loaded.

In the Game Boy Advance game, different colored Yoshis can be found in question blocks on regular stages after finding each colored Yoshi on Star Road. Cape Mario/Cape Luigi can find a blue Yoshi, Fire Mario/Fire Luigi can find a red Yoshi, and Super Mario/Super Luigi and Small Mario/Small Luigi can find a yellow or green Yoshi. In the SNES game, only green Yoshis can be found in question blocks outside of Star Road and the Special World.

Ghost Houses and Fortresses do not have Dragon Coins on the SNES version, but appear in every level in the Game Boy Advance version. Because of this change, some level designs were reconfigured. The Dragon Coins will also turn into Peach Coins (after a cutscene) if all five Dragon Coins are collected in every single level in the Game Boy Advance game.

In the SNES version, enemy changes and Autumn-colored levels appear after completing the Special World. In the Game Boy Advance version, all 96 exits must be cleared instead.

Additionally, the Game Boy Advance version includes a cutscene showing Luigi in a balloon tossing out masks to the Koopas after unlocking the Autumn-colored levels, whereas the changes in the SNES version are first seen when entering Yoshi's house upon exiting the Special World.

Galoombas and Pokeys also have different appearances in the Game Boy Advance version, along with Koopas, Bullet Bills and Piranha plants like the SNES version.

In the SNES game, when Mario or Luigi gets hurt while having a power-up (i.e. fire flower or cape), Mario and Luigi will become Small Mario/Small Luigi, as they did in Super Mario Bros. In the Game Boy Advance game, Power-up Mario/Power-up Luigi will become Super Mario/Super Luigi when they are hurt, as they do in Super Mario Bros. 3. In both versions, if Super Mario/Super Luigi get hurt, the reserve item in the box at the top of the screen will drop down.

In the SNES version, the "Top Secret Area" stage is a yellow dot on the map. In the Game Boy Advance version, a smiling bush represents the stage.

In the SNES game, the timer for Bowser's Castle is 400 seconds; there is no time limit when fighting Bowser. In the Game Boy Advance game, the timer was increased to 800 seconds if entering through the front door and 600 seconds if entering from the back door due to the timer continues when fighting Bowser.

In the SNES game, the "Top Secret Area" stage does not have a timer. In the Game Boy Advance game, the "Top Secret Area" has 200 seconds on the timer.

A status menu is accessible on the world map by pressing the Select button on the Game Boy Advance version. The status menu allows the player to see total play time, score, the number of exits found by each brother, whether Princess Peach has been saved, and whether all of Dragon coins have been collected.

After completing all exits on every level, a stage select screen will also become available that will take the player to any chosen level.

In the SNES game, the maximum number of lives a player could have is 99. In the Game Boy Advance game, this was changed to 999.

The international version has several changes to levels when compared to the Japanese release. These include minor graphical changes to fix mistakes or to provide more obvious information to the player, along with several changes to make the game slightly easier.
These changes are also present in the Game Boy Advance release.

The game contains an unused sprite of the word "and", it's not known exactly where this would have appeared, but it was most likely intended to be used as part of the copyright text.

In the SNES release, Yoshi's house has seven berries on it. In the Game Boy Advance release, there are ten berries.

The sleeping fish enemy, known as "Rip Van Fish", will typically chase down Mario upon being woken up. However, if Mario is under the effect of a star power-up, Rip Van Fish will make an effort to flee in the opposite direction of Mario instead.