Lonesome Cowboys prominently features multiple openly gay characters and same-sex relationships among its cowboy ensemble. The film portrays these dynamics with a non-judgmental, uninhibited, and often playful tone, presenting queer identity and desire as a natural aspect of the characters' lives without condemnation or ridicule. The net impact is affirming, showcasing dignity and agency.

The film features Ramona, a transsexual character, as part of its ensemble. Her identity is presented matter-of-factly within the film's experimental and observational style, contributing to its exploration of gender and sexuality without a strong narrative focus on affirming or denigrating trans identity. The portrayal is neither overtly positive nor negative.