Herpes simplex virus (HSV) infection occurs worldwide but its epidemiology varies between different countries and between groups of individuals. Seroprevalence of HSV-1 and HSV-2 antibodies is a more accurate method of determining epidemiology of this infection. In this cross-sectional study, 467 blood samples were obtained from women attending the genitourinary medicine clinic (GUM) and antenatal clinic (ANC) in Middlesbrough, England. Analysis of their blood samples showed that 69.5% GUM patients had HSV-1 antibodies and the figure was 54.5% in the ANC patients, p < 0.0001, with odds ratio 1.9 (95% CI 1.30 to 2.78) for higher prevalence in the GUM group. Also, for HSV-2 antibodies, the seroprevalence was 21.6% for GUM and 8.1% for ANC patients, p < 0.0001, with an odds ratio of 3.13 (95% CI 1.70 to 6.02) for higher prevalence in the GUM group. The overall prevalence for HSV-1 is 63.2% and for HSV-2 is 15.8%. Many of these patients were asymptomatic and constitute a potential source for spreading the virus. Knowledge of HSV seroprevalence can help in planning sexual health promotion strategies.