Although studies on HIV knowledge have previously been conducted in central Serbia and southern Kosovo, none has included the Serbian population residing in the northern part of the Kosovo province. The aim of this study was to assess HIV-related knowledge and to estimate factors contributing to a higher HIV-related knowledge level among Serbian university students in the Kosovo province. A cross-sectional study including 1017 first- and fourth-year students enrolled at the University of Pristina temporarily seated in Kosovska Mitrovica was carried out during the academic year 2013–2014. The students completed a 31-item questionnaire comprising demographic data, HIV transmission knowledge and attitude towards HIV testing. Data were statistically analysed. Students demonstrated good knowledge of HIV (average 32.8 ± 3.3 out of a maximum 42). Only 5% of the students reported having been tested for HIV. Factors associated with being more knowledgeable about HIV were studying health-related disciplines (β = −0.09; 95% confidence interval [CI] −0.13, 0.00), using the internet as a source of information about HIV (β = −1.09; 95% CI −1.65, −0.52), having a positive attitude towards HIV testing (β = −0.43; 95% CI −0.59, −0.26), having a low self-perceived risk for HIV infection (β = 0.41; 95% CI 0.23, 0.56) and the position that one would keep the same level of contact with an HIV-positive person after learning their HIV status (β = 0.38; 95% CI 0.21, 0.55). Setting up specialized classes on this topic at high schools and universities could help to increase the awareness of HIV infection and promote HIV testing and a positive attitude towards HIV-positive persons.