Information on age- and gender-specific prevalence of herpes simplex virus (HSV) types 1 and 2 infections is crucial to guide genital herpes control strategies. Such data are not available from the newly independent Eastern European countries. We present of study of the age- and gender-specific prevalence of HSV-1 and HSV-2 in low risk populations in Estonia. Serum samples from 1016 children, 794 first trimester antenatal women, and 1036 blood donors (462M, 574F) were tested for HSV IgG antibodies by type-specific HSV-1 and HSV-2 assays. High seroprevalence rates of HSV-1 among children, pregnant women and (non-paid) blood donors were found. HSV-2 infection was not detected among boys. Gender differences in HSV-2 seroprevalence rates among people of reproductive age were observed: higher rates were recorded among pregnant women (23%) and female blood donors (21%), compared to 11% among male blood donors. HSV-1 seroprevalence was high in adults. HSV-2 seroprevalence was higher among females than males and increased substantially with age. HSV-2 prevalence in these non-high risk populations was relatively high, and may indicate a risk for an impending sexually- and STI- driven HIV epidemic.