In 2003 and 2005, the Thailand Ministry of Public Health-U.S. Centers for Disease Control and Prevention Collaboration and its partners conducted surveillance of human immunodeficiency virus (HIV) prevalence and risk factors among populations of men who have sex with men (MSM) in Thailand. In 2003, the assessment was conducted in Bangkok among a sample of MSM. In 2005, in addition to Bangkok, the assessment was conducted in Chiang Mai and Phuket provinces, and participants were categorized as MSM, male sex workers (MSW), or transgendered persons (TG). This report compares HIV prevalence among MSM in Bangkok during 2003 and 2005, reports HIV prevalence among the three populations in 2005, and summarizes the results of univariate and multivariate analysis of risk factors for HIV infection in 2005. The results indicated a significant increase in HIV infection among MSM in Bangkok from 2003 to 2005. The findings also indicated that in 2005, HIV infection was widespread among MSM, MSW, and TG in the three study locations. Moreover, the following risk factors were independently associated with HIV infection: being recruited from Bangkok or Chiang Mai (MSM), older age (MSM and TG), being recruited from a park or street location (MSW and TG), drug use (MSM), self-reporting a history of sexually transmitted infections (MSW), and self-reporting a previous HIV-positive test result or refusing to disclose a previous HIV test result (MSM and MSW). Sex with women during the preceding 3 months was inversely associated with HIV prevalence among MSW. More effective behavioral and biomedical interventions for MSM, MSW, and TG are needed to stop the spread of HIV in these populations.