Hepatitis E virus (HEV) infection is endemic in many developing countries and becomes of interest in the developed countries. Several animals are sources of HEV infection to humans. Recently, HEV was detected in the milk of cows in China, this data comes up with the probability of HEV transmission to humans via ingestion of contaminated milk. In Egypt, contaminated water and residing in rural communities are risk factors for HEV infection, while limited data is available on the zoonotic HEV transmission. Since pigs, wild boars, camels are not common in Egypt, we investigated if cows and/or cow milk represent a risk factor for HEV transmission in the Assiut governorate. Milk samples (n = 480), collected from Assiut city and 12 non-mixed dairy farms distributed in the rural communities, were tested for HEV markers such as anti-HEV IgG, HEV RNA, and HEV Ag. All milk samples collected from Assiut city (n = 220) were negative for HEV markers. Also, milk samples collected from 11 farms (n = 220) were negative for HEV markers. While, in one farm, we could detect anti-HEV IgG in 8 out of 40 samples (20%), HEV RNA and HEV Ag were detectable in 1 out of 40 samples (2.5%). However, we could not detect the HEV markers in the stool from anti-HEV IgG positive cows. Surprisingly, phylogenetic analysis of the isolated virus revealed it belonged to HEV-3 subtype 3a. Importantly, when cows from the positive farm were retested 1 month later, we observed an increase in the number of animals that were positive for anti-HEV IgG (10/40, 25%). In addition, the level of anti-HEV IgG was significantly higher in the milk of these cows in the second collection than the samples of the first collection suggesting ongoing infection on this farm. In conclusion: we reported that HEV-3 and/or HEV like agent was detected in the milk of the cow distributed in rural communities of Assiut governates. Investigation of the cow milk should be done to assess if the cow milk is a risk factor for HEV transmission for Egyptian people, especially in rural communities.