OBJECTIVE: To investigate the HIV incidence and its related factors among female sex workers (FSWs) in a high prevalence area where injection drug use is also widely documented. METHOD: A cross-sectional study of 1642 female sex workers (FSWs) was conducted in Honghe Prefecture of Yunnan Province. Interviewed-questionnaires were administrated to collect information on sexual partnerships, condom use and illicit drug using behaviors etc. Blood samples were collected to test for HIV antibodies, and all HIV seropositive specimens were tested with the BED IgG capture-based enzyme immunosorbent assay (BED-CEIA) to distinguish between new and established HIV infection (<153 days). RESULTS: 15.9% (261/1642) of participants reported ever having used drugs, and 7.4% had injected in recent 3 months. The overall HIV prevalence was 10.2% (168/1642), among which 16.7% (28/168) were identified as recent infections using BED-CEIA. The crude HIV incidence estimated from BED-CEIA results was 4.4 (95%CI 2.8-6.0) /100 person years (PY). Multivariate logistic analysis showed that an illicit drug using history (by either self-reporting or urine opiates testing) was both significant risk factors both for HIV established and recent infection (each p<0.05). Drug using FSWs (DU-FSW) reported more male clients in the previous week, and had significantly higher prevalence of HIV, chlamydia trachomatis and HSV-2 as compared to non DU-FSW (each p<0.05). CONCLUSION: Our results show that a history of drug use poses significant risks for both new and established HIV infection among FSWs, and that HIV-incidence among Honghe FSWs is relatively high compared to similar populations. Comprehensive interventions targeted at DU-FSWs' injection drug using and high risk sexual behaviors are urgently needed to reduce the rapid spread of HIV epidemic.