Hepatitis E virus (HEV) is the leading cause of human enterically-transmitted viral hepatitis occurring around the world both as outbreaks and as sporadic cases. Non-human primates (NHPs) have been experimentally infected with HEV, but few studies have been reported about natural infection in wild-living and zoo monkeys. In order to provide a more complete picture on the epidemiology of HEV in NHPs living in controlled environment, we investigated the presence of HEV by screening serologically and molecularly a historical collection of 86 sera from seven different species of primates housed at the Zoological Garden (Bioparco) of Rome, Italy. By using an enzyme-linked immunosorbent assay based on the recombinant capsid protein of a Gt3 HEV strain, IgG antibodies were detected in three macaques (4.8%; 3/62) and in a white-crowned mangabey (16.6%; 1/6), with an overall prevalence of 4.6% (4/86). This positivity was confirmed when assessed the sera by western blotting. Rescreening the sera for IgM and viral RNA, all the samples resulted negative. Also, HEV RNA was not found when 17 stool samples were analyzed by RT-PCR. Although these results suggest that none of the monkeys housed at the Bioparco of Rome in the 17-year time frame spanning 2001 to 2017 developed acute or at least sub-acute HEV disease, the detection of IgG antibodies demonstrated that animals living in this setting were exposed to HEV or to antigenically related viruses.