The hepatitis E virus (HEV) has been described in humans and various animal species in different regions of the world. However, the knowledge on natural HEV infection in non-human primates and the corresponding risk of zoonotic transmission is scarce. To determine whether primates in captivity are affected by HEV infection, we investigated 259 individual sera of clinically healthy non-human primates of 14 species from nine German zoos. Using a commercial double-antigen-sandwich ELISA and a commercial IgG ELISA, 10 animals (3·9%) reacted positive in at least one assay. Three ape species and one Old World monkey species were among the seropositive animals: bonobo (Pan paniscus), gorilla (Gorilla gorilla gorilla), lar gibbon (Hylobates lar) and drill (Mandrillus leucophaeus). Testing for anti-HEV-IgM antibodies by commercial ELISA and for viral RNA by reverse-transcription real-time polymerase chain reaction resulted in negative results for all animals indicating the absence of acute HEV infections. In the past, no clinical signs of hepatitis were recorded for the seropositive animals. The results suggest that non-human primates in zoos can get naturally and subclinically infected with HEV or related hepeviruses. Future studies should evaluate potential sources and transmission routes of these infections and their impact on human health.