Hepatitis E is an important public health concern disease that causes acute hepatitis in humans with different hepatitis E virus strains reported among domestic and wild animals. In Lithuania, seroprevalence studies on animals considered to be possible virus reservoirs, particularly in wildlife species, had never been investigated before. In order to assess the actual distribution of the virus in the domestic pig and wildlife species, serum samples originating from different geographic areas of Lithuania were tested for the presence of antibodies to the virus using enzyme-linked immunosorbent assay. The study results proved that hepatitis E virus infection is prevalent among domestic pigs, wild boar, moose, and roe deer in Lithuania, with the overall prevalence of IgG antibodies against hepatitis E virus being 43.75%, 57.05%, 11.76%, and 1.20%, respectively. Significantly higher (P < 0.01) seroprevalence values of 53.66% and 80% were observed in weaned pigs and adult wild boar. Herds with 15 001–30 000 pigs were 2.4 times more likely (P < 0.01) to test positive for hepatitis E virus antibodies (70.18%), than herds with ≤ 500 pigs (21.11%). The differences in seroprevalence rates between domestic pigs (from 25 to 66.67%) and wild boar (from 42.86 to 73.33% ) in all 10 counties were not significant (P > 0.05). Estimated low seroprevalence rate in moose, roe deer and absence of antibodies in red deer suggest that such wildlife species are accidental hosts of hepatitis E virus or at least hepatitis E virus is not present in Lithuanian cervides.