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Mario is the eponymous main characters Sheldon's Game.
Sheldon played Super Mario Brothers when he was a child, the only happy time he could remember in his childhood. Amy played the theme music during dinner at her apartment in "The Launch Acceleration" to make him happy and possibly draw them closer together.
- In "The Electric Can Opener Fluctuation", Howard sits down at the lunch table with a new moustache. He says he calls it "the Clooney", while Raj replies "I call it the Mario and Luigi, but whatever."
- In "The Codpiece Topology", Sheldon's mother sends him his Nintendo 64 along with Super Mario 64. He is later seen playing it on his laptop, using a "poorly coded Nintendo 64 emulator." He even says "Hop, you little plumber. Hop, hop, hop!" and "Everybody has a date. Even you, Mario, going after Princess Peach. And what am I doing? I'm just enabling you."
- In "The Gorilla Experiment", Raj and Sheldon are playing Mario Kart Wii with the Wii Wheel. Raj is shown playing with Yoshi on a kart and Sheldon playing as Mario on a bike in Mario Circuit. Raj wins and says to Sheldon, "Face it, dude, whether it's a real car or a virtual cartoon car, you can't drive." Sheldon says it was only because he hit a tree, and Raj adds, "And a cow and a penguin" (hinting that they were also racing on Moo Moo Meadows and Sherbet Land). Raj tells him he really needs some cheat codes, motor skills, and a genie who grants wishes to little boys who suck at Mario Kart
- In "The Bozeman Reaction", Leonard's and Sheldon's apartment is robbed. Several consoles and games are mentioned as being stolen. The following are Mario-related: Super Mario Bros., Super Mario Galaxy, and Mario & Sonic at the Olympic Winter Games. When writing in a journal in bed, Sheldon also mentions, in his thoughts, that "outside, evil lurks, probably playing Donkey Kong on my classic Nintendo.
- In "The Staircase Implementation," Raj, Howard, and Leonard are seen playing a Nintendo GameCube game on the couch. The music and sounds emitted from the TV tell that they are playing Mario Kart: Double Dash!!, more specifically Waluigi Stadium or Wario Colosseum.
- In "The Shiny Trinket Maneuver," Howard, Raj, and Leonard are, in one scene, playing Donkey Kong Jenga. During this scene, Howard is talking to Raj and Leonard about him and Bernadette possibly not having children and states that "even Donkey Kong had Donkey Kong Jr." He also mused about how Donkey Kong would have taught his son how to "kidnap princesses and throw barrels at Italian plumbers", but this is actually an erroneous observation as Donkey Kong's damsel-in-distress, Pauline, was not a princess.
- In "The Launch Acceleration", during Amy and Sheldon's date at her house, part of Amy's attempt to make him love her more includes playing the Super Mario Bros. ground theme as "romantic music." Almost immediately after, Sheldon admits that playing the game was the best 600 hours of his life.
- In "The Itchy Brain Simulation", Leonard attempts to return a long-overdue copy of the Super Mario Bros. movie.
- In "The Indecision Amalgamation", when Sheldon and Amy are at Best Buy contemplating whether to buy a PlayStation 4 or Xbox One, several advertisements for Mario games can be seen in the background. These include Donkey Kong Country Returns 3D, Super Mario 3D World, Mario Party: Island Tour, and Mario & Luigi: Dream Team.
- In "The Misinterpretation Agitation", during the scene when Dr. Lorvis invites Leonard, Sheldon, Howard, and Raj to see his collection of various types of memorabilia, arcade cabinets of Donkey Kong and Mario Bros. can be seen among them. Leonard is later seen playing Donkey Kong at the end of the episode.