Viral hepatitis is one of the major problem worldwide. The detection of hepatitis E virus (HEV) antibodies IgM (HEV-IgM) and HEV-RNA in donated blood in Aseer Region (Southern part of Kingdom of Saudi Arabia) to detect its prevalence was attempted. The study was conducted on random blood samples collected from healthy blood donor volunteers, who were referred to blood transfusion centers found at Aseer region, during the period from March 2012 to January 2013. All the collected blood units were screened for HEV-IgM, hepatitis B surface antigen (HBsAg), antibodies to HB core antigen (HBcAb), hepatitis C virus (HCV), human immunodeficiency virus 1 and 2 (HIV-1/2), human T-cell lymphotropic virus (HTLV) I/II, venereal disease research laboratory (VDRL), and malaria. All donated blood samples were checked for HBV-DNA, HCVRNA, and HIV-RNA by nucleic acid test (NAT) technology. Of 7267 donors (26 females (0.36%) and 7241 males (99.64%)) blood donors screened, with median age of 28 (female) and 30 years (males), 10 (0.13%) were HEV-IgM border-line but negative by RT-PCR. In conclusion, prevalence of HEV-IgM in blood donors at Asser region is zero. Vaccination program against HEV is still needed to prevent future outbreaks. Further studies are warranted to determine the true seroprevalence of the virus in the society at large...