This study reports trends in HIV-1 incidence in Cambodia among different target groups in the HIV-1 Sentinel Surveillance Program in 1999, 2000, and 2002, using the newly developed IgG capture BED-enzyme (HIV subtypes B, E and D) immunoassay (BED-CEIA). HIV-1-positive specimens (n=3599) from 4 sentinel groups in the HIV-1 Sentinel Surveillance Program from 1999 to 2002-brothel-based commercial sex workers (CSWs), indirect commercial sex workers (IDSWs), police, and women attending antenatal clinics (ANCs)-were tested using the BED-CEIA. Annualized incidence rates were calculated for each group and each geographic region. Between 1999 and 2002, incidence rates declined among CSWs from 13.9% to 6.45%, among IDSWs from 5.92% to 2.87%, and among police from 1.58% to 0.26%. In the ANC group, the incidence remained stable, 0.64% in 1999, 1.11% in 2000, and 0.59% in 2002. However, there was an increasing trend among ANCs in rural areas, from 0.12 to 0.89%. In conclusion, HIV-1 incidence among CSWs, IDSWs, and police has declined between 1999 and 2002; however, the incidence has not declined in the ANC group.