The prevalence of anti-hepatitis E virus (HEV) antibodies has a high heterogeneity worldwide. South American data are still scarce. The aim of this study was to evaluate the prevalence of HEV in populations at risk in comparison to blood donors (BD). A cross-sectional study was carried out in adults of different risk populations including crack users (CK), residents in a low income area (LIA), cirrhotic (CIR) and liver transplant patients (LT) compared with BD. The WANTAI HEV ELISA test was used and real-time PCR (in-house for screening and ALTONA as confirmatory test) for HEV RNA screening. A total of 400 participants were included. Anti-HEV IgG was positive in 19.5% of the total sample, reaching the highest rate in the CIR group, 22.5%, followed by CK, LT, and LIA (20%, 18.7%, and 17.5%, respectively). The prevalence found in BD individuals was of 18.7% (p = NS). Anti-HEV IgM was positive in only 1.5% of the sample (6/400). No blood or stools samples were positive for HEV RNA. The seroprevalence reported is among the highest rates ever found in Brazil. Considering the intense diagnostic investigation, data show that HEV circulation is more common that might be expected in our country.