Herpes simplex virus type 2 (HSV-2) can contribute to the spread of HIV. From March 2006 to November 2009, female sex workers (FSWs) in Yunnan, China were recruited into an open cohort study to determine incidence and risk factors for HSV-2 acquisition. Participants were interviewed and tested for HSV-2 and other sexually transmitted infections (STIs) every six months. A multivariate Cox proportional hazards regression model with time-dependent variables was used to measure associations with HSV-2 acquisition. In 3.5 years, 83 incident cases of HSV-2 infection were diagnosed, yielding an overall incidence of 21.9 per 100 person years (PY) (95% confidence interval [CI], 17.8-26.3). Working in higher risk commercial sex venues, current Neisseria gonorrhoeae infection, age of sexual debut <18 years and lack of a regular sex partner were independent factors associated with HSV-2 acquisition. The high incidence of HSV-2 suggests that prevention methods for HIV/STI control are urgently needed.