Students at the University of Botswana, an at-risk group, have previously been shown to have high levels of risky sexual behavior despite widespread knowledge that these behaviors might lead to HIV-1 infection. As there have recently been considerable efforts focused on HIV-1 prevention in Botswana through nationwide media education campaigns and the opening of voluntary counselling and testing centers, re-evaluation of HIV-related knowledge, attitudes, and practices among students is needed. A cross-sectional survey was administered to 393 students chosen via a random cluster method. Respondents were 50% junior and 50% senior students with 42% males. Half (52%) were “single”, 44% were “in a relationship”, and 4% were “married”. The mean percentage of knowledge questions answered correctly was 96%. 98% agreed that all sexually active adults should know their status and that condom use is important, but only 56% believed getting tested was common and 66% believed that it was common for students to always use a condom. As with the previous survey, we again found that students had excellent knowledge yet perceived use of testing services and condoms remain lower than might be predicted based on knowledge scores.