Studies have shown that the level of knowledge that the general population or certain social and professional groups have about homosexuality can predict prejudice based on sexual orientation and homophobic attitudes. This exploratory study aimed to evaluate Romanian university students’ knowledge about homosexuality. A survey was conducted among 535 subjects enrolled in bachelor’s and master’s degree, as well as pre-service teacher training programs. The mean age of participants was 24.82 years (range 18-52; SD = 8.08). The sample included 432 females and 103 males. Participants completed a 15-item questionnaire based on C. A. Koch’s modified version of The Knowledge About Homosexuality Questionnaire developed by M. B. Harris, J. Nightengale și N. Owen. For the entire sample, the mean number of correct answers was 5.48 (range 0-13; SD = 2.72) out of 15 items, i.e., 36.5%. This result indicates a relatively modest level of knowledge about homosexuality in the targeted population. Significant differences were observed depending on the study program, orientation on the axis of conservatism vs. liberalism, casual social contact and friendship with a lesbian/gay person. Replication of the survey on a more representative sample of unversity students in terms of gender, age and majors is needed. However, our investigation indicates the need for university students to be knowledgeable about psychological, social, and legal issues affecting lesbian and gay people, in order to better address and evaluate their opinions and personal feelings surrounding the topic of homosexualiy as a whole.