Pathogenic caliciviruses of the genus Vesivirus circulate in oceanic ecosystems and spread to and among terrestrial mammals. Isolation of Vesivirus from natural and laboratory infections in humans led to this investigation of Vesivirus seroprevalence and viremia. Sera from four groups were tested for antibodies to Vesivirus as follows: blood donors whose units were cleared for donation, blood donors whose units were not accepted for donation solely because of elevated blood liver alanine aminotransferase (ALT) concentrations, patients with clinical hepatitis of unknown but suspected infectious cause, and patients with clinical hepatitis of unknown cause but associated with blood transfusion or dialysis. Additionally, sera were tested for Vesivirus genome by three methods: dot‐blot and two reverse transcription‐polymerase chain reaction (RT‐PCR) methods. The calculated seroprevalence against Vesivirus virions within these groups (N = 765) was 12%, 21%, 29%, and 47%, respectively (P < 0.001 for group differences). Additionally, 11 (9.8%) of 112 sera tested yielded RT‐PCR amplicons that by nucleotide sequence were distinct from each other and related to known Vesivirus. These data indicate that some blood donors in the population tested have serologic evidence of previous Vesivirus infection and some also have Vesivirus viremia. These results justify further investigation of an association between Vesivirus infection and illness in humans. J. Med. Virol. 78:693–701, 2006. © 2006 Wiley‐Liss, Inc.