The goal of this research was to examine the relationship between gender role attitudes, gender identification and negative attitudes toward gays and lesbians. Research was conducted over the internet on 1167 prticipants, 336 male participants and 831 female participants. The questionnaire examined attitudes toward gays and lesbians (Herek, 1987 ATLG scale – Attitudes Toward Lesbians and Gay Men Scale), gender role attitudes (Attitudes toward gender role equality scale - Kamenov, Huić, Jugović; 2011), gender identification (a scale was constructed for this research, based on exiting questionnaires). Results have shown that men hold more negative attitudes toward gays and lesbians than women, and that both men and women hold more negative attitudes toward gays than lesbians. Also, it has been shown that gender role attitudes and gender identification are significantly related to attitudes toward gays and lesbians, and that gender role attitudes have significant contribution in predicting attitudes toward gays and lesbians. Results were considered from the gender perspective.