Introduction: Herpes simplex virus (HSV) is the main co-factor for heterosexual transmission of the human immunodeficiency virus (HIV) in sub-Saharan Africa, and could be involved in the dynamics of the HIV epidemic in Senegal. Methodology: Genital shedding of HSV was evaluated in adult females who had visited the provincial healthcare centres in Diass, Louga, and Kebemer in Senegal. Study subjects were interviewed by a healthcare worker for sociodemographic characteristics and sexual behavior, and HIV serology was offered. In addition, cervical secretion lavage samples were evaluated for HSV DNA by real-time polymerase chain reaction (PCR), the melting curve analysis of which permitted distinction between HSV type 1 (HSV-1) and HSV type 2 (HSV-2). Results: Among 302 women (mean age, 40 years) enrolled, none were infected by HIV. The mean age at first sexual intercourse was 20 years, and the mean number of sexual partners in the previous year was 1.3 (range, 1-7). Only 6 of 302 (1.9%) women had cervico-vaginal secretions positive for HSV DNA. No association between HSV DNA shedding and any sociodemographic or biological variables was found. Surprisingly, genital shedding of HSV-1 was found in two (0.7%) women, representing 33% of herpes-shedding women, and HSV-2 in four (1.5%) women. Conclusions: Taken together, our observations indicate a low prevalence of HSV DNA genital shedding in adult Senegalese women.