My Hero Academia: Two Heroes does not feature any explicit LGBTQ+ characters or themes. The storyline centers on the main protagonists and new characters introduced for the film, with no discernible queer representation or related plot points.

My Hero Academia: Two Heroes does not feature any identifiable transsexual characters or themes. The narrative is solely focused on its primary plot of heroics and villainy on I-Island, making the portrayal N/A.

The film features several female characters who participate in combat using their unique superpowers (Quirks) against male opponents. However, their victories are achieved through the application of these powers, such as acid attacks, sound waves, or gravity manipulation, rather than through direct hand-to-hand combat, martial arts, or melee weapon skills.

The film introduces new characters, but all established characters from the 'My Hero Academia' manga and anime retain their original canonical genders. No characters historically or widely established as one gender are portrayed as a different gender.

The film is an animated adaptation of the My Hero Academia manga/anime. All major characters are depicted consistently with their established racial and ethnic portrayals from the source material, with no instances of a character's race being changed.