Road Trip utilizes perceived or implied homosexuality primarily as a source of crude, uncomfortable humor and misunderstanding. The film's comedic approach often plays on discomfort and stereotypes, offering no counterbalancing positive or affirming portrayals of LGBTQ+ identity. The net impact is problematic and negative.

Road Trip features a transsexual character, Tiffany, whose identity is revealed as a punchline. The scene depicts a main character's disgust and subsequent humiliation, which is played for comedic effect. The portrayal reinforces negative stereotypes and contributes to a problematic view of transsexual individuals, lacking any dignity or positive representation.