The show features April O'Neil, a character confirmed by creators to be bisexual. While her identity is present, it is not explicitly explored or made a central theme within the narrative. Her portrayal is generally positive, but her sexual orientation is incidental to her on-screen character development and plotlines, resulting in a neutral net impact.

The show features female characters April O'Neil and Cassandra Jones who regularly engage in and win close-quarters physical combat. April uses her baseball bat, while Cassandra employs a hockey stick and hand-to-hand skills, to defeat multiple male opponents, including Foot Clan soldiers and various mutants.

April O'Neil, a prominent character consistently depicted as white in prior Teenage Mutant Ninja Turtles iterations, is portrayed as a Black teenager in this series, constituting a race swap.