Hepatitis A virus (HAV) has a worldwide distribution, although this distribution tends to be uneven among geographical regions and population groups. The prevalence of anti-HAV antibodies in the general population varies widely among countries. In Europe, the seroprevalence of HAV is reported to range from 32% to 88%. The aim of this study was to determine the seroprevalence of HAV among the general Croatian population. During a 2-year period (2008-2009), a total of 791 serum samples were tested for the presence of anti-HAV total (IgM+IgG) and anti-HAV IgM antibodies using an automated enzyme-linked fluorescent assay (Mini Vidas; bioMérieux, Marcy l'Etoile, France). The overall anti-HAV seroprevalence was 41.6%. The observed difference in the seroprevalence rates among male and female patients was not statistically significant (44% vs. 39.6%, P = 0.218). A marked increase in anti-HAV seropositivity with age was observed (P < 0.001). The seroprevalence did not differ significantly between participants residing in rural regions (45.3%) and those residing in urban regions (40.6%, P = 0.292). Our results corroborate those of seroprevalence studies in other developed countries. More than half of the Croatian population (59.4%) is susceptible to HAV infection. Older age is an important predictor for being anti-HAV positive.