Both Mach Rider's identity and gender are ambiguous. In the original game, the Mach Rider is portrayed with a masculine muscular build. The NES and Famicom manuals constantly refer to the Mach Rider by name ("the Mach Rider") or as "you" from the player's perspective, and do not use specific pronouns throughout, though the NES manual does at one point break from this to call Mach Rider a "he". However, the arcade port Vs. Mach Rider's stage clear screens slowly introduce piece-by-piece an image of a skimpily-dressed woman (bearing similarities to Jane Fonda's appearance in the 1968 film Barbarella) holding a dagger. It has never been specified if this character is the Mach Rider and is meant to be a twist reveal in a similar vein to Samus from Metroid, or if this is one of the other wasteland survivors. Years later, Mach Rider's trophy description in both the English and Japanese versions of Super Smash Bros. Melee years use he/his as pronouns. Even later on, Captain Rainbow, a Japan-only game centered around obscure and neglected Nintendo heroes, has an unused model for the Mach Rider being depicted with a skinny feminine waist, while not having visible breasts due to wearing armor.

When Birdo (known in Japanese as Catherine) was first introduced in Yume Koujou: Doki-doki Panic, the game's manual gave this description:


This carried over to the manual for the game's Western reskin as Super Mario Bros. 2 (known as Super Mario USA in Japan), where it instead states that Birdo's preferred name is "Birdetta". Starting with the commercial for Super Mario USA, Birdo was depicted in Japanese media as a flamboyant and flirtatious okama (a Japanese-language derogatory slur used to refer to gay men, transgender women, male crossdressers, and/or effeminate men) in both her voice acting and mannerisms for comedic purposes. However, this depiction, as well as the Western Birdetta name, were largely omitted from English localizations of future Mario games, with some exceptions:

• Mario & Luigi: Superstar Saga, where Birdo appears as an accomplice of Popple, who feels confused and discomforted by her.
• Super Smash Bros. Brawl, where Birdo's trophy description calls Birdo "gender indeterminate", uses "it" instead of a gendered pronoun, and includes the omitted Birdetta name.
• Super Mario Party, where Birdo is referred to with she/her pronouns in the American English script, he/him pronouns in the British English script, and no gendered pronouns in the Japanese script.

Birdo's Japanese depictions drew characteristics from drag queens and transgender women in Japanese pop culture, and Birdo would retain toned down elements of these in subsequent reappearances. Birdo frequently refers to herself in Japanese media with both the first-person feminine pronoun "atashi" and the gender-neutral first-person pronoun "watashi", invoking the latter in a feminine manner. In the Satellaview game BS Super Mario USA, the three types of Birdo are voiced by Shiroi Heya nightclub performers Jun Donna, Rika, and Akemi, all of whom identified as newhalves (a Japanese term interchangeably used to refer to transgender people and male crossdressers). Birdo's gender is most explicitly addressed in Captain Rainbow, where she is depicted again as an okama and is arrested for using a women's restroom. This forms the basis for a sidequest where she can only be exonerated once the player finds a censored item in her house called the "female proof" that is strongly implied to be a vibrator. In one trailer for the game, Little Mac describes Birdo as a "young lady", before pausing and restating: "Depending on one's view point, she is a young lady."

In 2015, a representative for Nintendo told the Japanese news site Netorabo that Birdo, as well as Yoshi, were neither male or female, or even dinosaurs; Birdo was "someone who believes they are a girl [...] 'Yoshi' is 'Yoshi' and 'Catherine' is 'Catherine'".