Brazil has been classified as moderately endemic for hepatitis E virus (HEV) infection. However, data on the seroprevalence of HEV in this region are limited. This study evaluated the prevalence of past or present HEV infection among blood donors in the metropolitan area of Itajai Valley, Southern Brazil, a region of predominant German heritage, where cultural habits result in a high consumption of pork. Serum samples from 300 blood donors were tested in December 2014 using serological and molecular methods. Anti-HEV IgG antibodies were detected in 30 (10%) subjects, and categorized age groups revealed an age-dependent increase of HEV seroprevalence. Only one subject had anti-HEV IgM, whereas none tested positive for HEV-RNA. The present data demonstrate a higher seroprevalence of anti-HEV IgG in blood donors than previously reported in Brazil. J. Med. Virol. 88:361–364, 2016. © 2015 Wiley Periodicals, Inc.