The aim was to study the type-specific seroprevalence of Herpes simplex virus (HSV)-1 and HSV-2 infections and the associated risk factors in children, adolescents and adults in Greece. A total of 1867 serum samples from children, adolescents and adults of both genders aged from 0 to 60 years were collected from three large hospital-referral centres in Athens. All sera were tested for type-specific antibodies to HSV-1 and HSV-2 using HerpeSelect IgG ELISA tests (Focus Diagnostics Cypress, Cal, USA). Overall age-adjusted seroprevalence of HSV-1 and HSV-2 was 72.0% and 10.2%, respectively. HSV-1 seropositivity was low in children up to nine years of age, increased sharply in adolescence, and was higher in females than males in each group surveyed. HSV-2 seropositivity was low (0.8%) below 20 years of age and increased to a maximum prevalence of 18.7% in men and 22.7% in women. Multivariate risk factor analyses indicated that HSV-1 seropositivity was associated with socioeconomic indicators (e.g. lower educational level, residency outside greater Athens), whereas HSV-2 was associated with sexual behavioural factors (e.g. being divorced, greater number of lifetime sexual partners). HSV-2 risk factor profiles were similar in women and in men. This first large seroprevalence study in Greece showed a high age-standardized HSV-1 seropositivity after adolescence and a relatively low age-standardized HSV-2 seroprevalence from birth to 60 years of age. Dual seropositivity to HSV-1 and HSV-2 was low (0.6%) in females under 20 years of age, suggesting that the potential use of an HSV-2 prophylactic vaccine in adolescents could reduce the spread of HSV-2 infection.