The objectives of this cross-sectional study were to assess the prevalence and identify predictors of HIV-1 and HIV-2 infection among students in three cities of Mali. Between January and June 2005, we assessed HIV knowledge, attitudes, associated sexual behaviors and tested HIV serostatus among 950 high school and university students in Sikasso, Bamako, and Koulikoro cities, using a combination of enzyme-linked immunosorbent assay (ELISA) and Western blot testing. Univariate and logistic regression analyses were used to determine predictors of infection among students. Mean HIV prevalence was 3.1%, ranging from 1.8% in Sikasso to 3.6% in Bamako. The results showed the presence of all three HIV subtypes in Bamako, though HIV-1 predominated in all cities. Infection rates were slightly higher among males (3.6%) than among females (2.8%), but the difference was not significant. The single significant predictor of HIV infection was knowledge of HIV routes of transmission (p=0.01). HIV prevalence rates observed in this population were higher than general adult prevalence rates previously reported for Mali. HIV/AIDS education and prevention campaigns should be targeted to students at both the secondary and university levels. Students may represent an informative HIV sentinel population for Mali.