Hepatitis E virus (HEV) is a zoonotic pathogen of which several species of animal were reported as reservoirs. Antibodies to HEV and HEV RNA have been detected in some Chinese population and swine groups but few other domestic animals. In this study, to investigate the HEV prevalence, we tested sera from 788 pigs, 100 cows, 50 goats, 49 horses, 101 pet dogs, 105 chickens, 47 duck and 45 pigeons in eastern China for anti-HEV immunoglobulin G (IgG). We also tested 50% of the swine sera, all of sera from the other domestic animals and 13 Shanghai human sera which were positive for anti-HEV immunoglobulin M (IgM) for HEV RNA using reverse transcriptase-polymerase chain reaction. Our results indicated that 82.5% (222/269) of the sows, 53.9% (104/193) of the 4- to 6-month-old swine, 63.4% (168/265) of the 1- to 3-month-old swine, 55.7% (34/61) of the slaughterhouse swine, 24% (12/50) of the goats, 16.3% (8/49) of the horses, 17.8% (21/101) of the pet dogs, 6% (6/100) of the cows, 12.8% (6/47) of the ducks, 4.4% (2/45) of the pigeons and 1.9% (2/105) of the chickens exhibited positive for anti-HEV IgG. Inhibition assay confirmed the infection with HEV or HEV-like viruses in these domestic animals except pigeons and chickens. From the sera, we isolated 18 swine HEV strains, one horse HEV strain and two human HEV strains. Sequence analysis showed that the horse HEV isolate and one swine isolate belonged to genotype 3. The other isolates belonged to genotype 4. The two human isolates were phylogenetically closely related to eight of the swine isolates. In short, the presence of anti-HEV antibody had been confirmed in several species of domestic animals in eastern China and HEV RNA has been identified in swine, human and horse. This suggested that the authorities should pay more attention to the prevalence of HEV in eastern China.