Female sex workers in Europe have low levels of sexually transmitted infections, attributable to condom use. The aim of this paper is to describe the seroepidemiology of HSV-1 and HSV-2 in female sex workers in London by using a 15-year prospective study of 453 sex workers. The seroprevalence of HSV-1 was 74·4% and independently associated with birth in a ‘transitional country’ (OR 5·4, 95% CI 1·61–18·20). The seroprevalence of HSV-2 was 60% and declined over time; it was also independently associated with time in sex work (OR 2·12, 95% CI 1·23–3·65) and birth in a ‘developing country’ (OR 2·95, 95% CI 1·34–6·48). We show that a cohort of sex workers with extensive condom use and little known sexually transmitted infection have high levels of HSV-1 and HSV-2 infection, suggesting that condoms may not be universally protective. Sex workers are candidates for HSV vaccine efficacy or intervention studies.