While past research has documented significant relationships between both athletic and Greek system (fraternity and sorority) membership and negative attitudes toward gays and lesbians, such work seems to vilify membership in athletics and the Greek system as causal mechanisms of homophobia. In this way, athletes and Greeks may be easy targets to blame for the homophobia evident on college campuses. However, it may be more productive to attempt to identify underlying constructs that are related to collegiate athletes’ and fraternity/sorority members’ attitudes to best inform campus LGBT programs. In this study, athletes’ and fraternity and sorority members’ attitudes toward lesbian, gay, bisexual men and women, and transgender (LGBT) individuals are examined. Further exploration considers how LGBT affiliations (i.e., knowing LGBT individuals, knowing about LGBT programs, and having Gay–Straight Alliances in high school) and religiosity may mediate the relationship between athletic and Greek membership and attitudes toward LGBT individuals. Results show that being a male athlete or being a fraternity member is related to unsupportive attitudes toward LGBT individuals; however, LGBT affiliations and religiosity mediate some of the significant relationships. In contrast, being a female athlete is unrelated to attitudes toward LGBT individuals while being a sorority member is related to attitudes toward LGBT individuals in some models. Campus LGBT program implications for both athletes and Greek system members are provided.