Abstract Background: Herpes simplex virus (HSV) infections are caused by 2 types of virus, HSV-1 and HSV-2. Both viruses are endemic worldwide. There are marked variations in the seroprevalences of HSV-1 and HSV-2 in Europe. The aim of this study was to determine the seroprevalence of HSV infections in Croatia. Methods: During a 3-y period (2008-2010), a total of 1672 patients were tested for the presence of HSV-1 and HSV-2 antibodies using an enzyme-linked immunosorbent assay. Results: The overall immunoglobulin G (IgG) seroprevalence rates were 72.5% for HSV-1 and 9.9% for HSV-2. There was no significant difference in seropositivity between males and females for HSV-1 (72.0% vs 73.0%) or HSV-2 (8.9% vs 10.7%). HSV-1 seroprevalence increased from 26.4% in those aged 6 months-9 y to 89.9% in those aged 40-49 y, and remained stable thereafter, ranging from 87.4% to 91.5% (p < 0.001). HSV-2 IgG seropositivity increased progressively from 5.7% in participants aged 20-29 y to 26.5% in participants aged ≥ 60 y (p < 0.001). HSV-1 seroprevalence did not differ between participants residing in urban and rural areas (72.5% vs 72.6%). Urban place of residence was a significant factor for HSV-2 seroprevalence in univariate analysis, but after standardization for age, it was no longer significant.Conclusions: Multiple logistic regression showed that age was a significant predictor of both HSV-1 and HSV-2 seropositivity, while female gender was a significant predictor of HSV-2 seropositivity. In pregnant women, obstetric history was not a significant predictor of either HSV-1 or HSV-2 seroprevalence.