Little is known about the HIV epidemic in conflict-affected southern Sudan. During 2002-2003, we conducted behavioral and biological surveillance surveys and sequential sampling in antenatal clinics in Yei, Western Equatoria, and Rumbek, Bar-el-Ghazal. HIV prevalence among individuals aged 15-49 years ranged between 0.4% in Rumbek town and 4.4% in Yei town, and among pregnant women between 0.8 and 3.0%, respectively. After the recent peace agreement, targeted prevention programmes are urgently needed to prevent further spread.