This is the last video game where Wario wears his original long sleeved shirt which was used in his early appearances. In all appearances after this game, he is seen wearing his short sleeved shirt.
This is the first game where Shy Guy receives a voice by Nate Bihldorff, who regularly voices Shy Guy in later Mario installments. This is also the first game where Takashi Nagasako provides his voice for Donkey Kong and later regularly reprises this role in later installments. Katsumi Suzuki begins his career as Diddy Kong's voice actor here as well.
One of Wario's Power Shots, the Ultra Hand Return, is based on a Nintendo toy invented by Gunpei Yokoi in 1966 called the Ultra Hand. The toy worked to grab objects from a distance, and Wario will use it to hit a ball that is out of reach.
In the intro is a tournament board with every character in the game. In the bottom left corner of the board you can see the team of Toad and Toadette, hinting that they were possibly planned to appear as playable characters at one point during development.