BACKGROUND: Yunnan is one of the provinces hardest-hit by HIV in China. To understand HIV epidemic dynamics and evaluate prevention effectiveness, we studied the changing trends in HIV-1 prevalence and incidence among five sub-populations in Yunnan. METHODS: Consecutive sentinel surveillances were conducted among people who inject drugs (PWID), male sexually transmitted diseases (STD) clinic attendees, and pregnant women for 2001-2010,female sex workers (FSWs) for 2007-2010, men who have sex with men (MSM) for 2008-2010. For the newly diagnosed HIV-seropositive samples, the recent infections were determined with BED-capture enzyme immunoassay (BED-CEIA), based on which HIV incidence was calculated for each sub-population using McDougal algorithm. RESULTS: From 231,117 individuals, 6,107 HIV-positive samples were tested with BED-CEIA, among which 964 samples were identified as recent infections. In PWID, HIV prevalence for 2001-2010 was between 27.16% and 18.35%, while the estimated incidence rate significantly decreased from 11.68% in 2001 to 1.70% in 2010. Among male STD clinic attendees, both the HIV prevalence (from 3.62% in 2001 to 1.73% in 2010) and incidence (from 1.10% in 2001 to 0.40% in 2010) showed a significant decreasing trend. In FSWs, the HIV prevalence for 2007-2010 kept stable (between 2.46% and 1.95%), while the HIV incidence significantly decreased (from 0.71% in 2007 to 0.31% in 2010). In MSM, the HIV prevalence (between 11.78% and 9.42%) and incidence (between 6.01% and 8.38%) remained stable at a relatively high level for 2008-2010. In pregnant women, the HIV prevalence (between 0.44% and 0.30%) and incidence (between 0.15% and 0.08%) remained stable for 2001-2010. CONCLUSION: The HIV incidences in PWID, male STD clinic attendees and FSWs showed the decreasing trend, supporting a positive effect of prevention strategies for these sub-populations. MSM with the highest HIV incidence have become the sub-population most at risk. In most sub-populations, the HIV prevalence did not decline, suggesting the disease burden is still heavy. These findings are valuable for developing HIV prevention strategies in Yunnan.