Autochthonous hepatitis E virus (HEV) infection is increasingly reported in industrialized countries and is mostly associated with zoonotic HEV genotype 3 (HEV-3). In this study, we examined the molecular epidemiology of 63 human clinical HEV-3 isolates in Canada between 2014 and 2022. Fifty-five samples were IgM positive, 45 samples were IgG positive and 44 were IgM and IgG positive. The majority of the isolates belong to the subtypes 3a, 3b, and 3j, with high sequence homology to Canadian swine and pork isolates. There were a few isolates that clustered with subtypes 3c, 3e, 3f, 3h, and 3g, and an isolate from chronic infection with a rabbit strain (3ra). Previous studies have demonstrated that the isolates from pork products and swine from Canada belong to subtypes 3a and 3b, therefore, domestic swine HEV is likely responsible for the majority of clinical HEV cases in Canada and further support the hypothesis that swine serve as the main reservoirs for HEV-3 infections. Understanding the associated risk of zoonotic HEV infection requires the establishment of sustainable surveillance strategies at the interface between humans, animals, and the environment within a One-Health framework.