We examined levels of, and reasons for, anti-gay and anti-lesbian prejudice (homophobia) in pre-service physical education (PE) and non-physical education (non-PE) university students. Participants (N = 409; 66% female; N = 199 pre-service physical educators) completed questionnaires assessing anti-gay and lesbian prejudice, authoritarianism, social dominance orientation (SDO), physical/athletic identity and self-concept, and physical attributes. ANCOVAs revealed that PE students had higher levels of anti-gay (p = .004) and lesbian prejudice than non-PE students (p = .008), respectively. Males reported greater anti-gay prejudice (p < .001), but not anti-lesbian prejudice, than females. Authoritarian aggression was positively associated with greater anti-gay (β = .49) and lesbian prejudice (β = .37) among male participants. Among females, higher authoritarian aggression and SDO was associated with greater anti-gay (β = .34 and β = .25, respectively) and lesbian (β = .26 and β = .16, respectively) prejudice. The physical identity-related constructs of athletic self-concept (β = .-15) and perceived upper body strength (β = .39) were associated with anti-gay attitudes among male participants. Physical attractiveness (β = -.29) and upper body strength (β = .29) were also associated with male participants' anti-lesbian prejudice. Regression analyses showed that the differences between PE and non-PE students in anti-gay and lesbian prejudice were largely mediated by authoritarianism and SDO. The present study is the first to examine the relationship between investment in physical/sporting identity and attributes and anti-gay and lesbian prejudice in PE/sport participants. In the present sample, anti-gay and lesbian prejudice was greater in pre-service PE students than non-PE students, but these differences appear to be explained by differences in conservative ideological traits. Additionally, physical identity and athletic attributes based around masculine ideals also appear to contribute to this prejudice in males.