Viral hepatitis is one of the major problem worldwide. The detection of hepatitis B virus (HBV) surface antigen (HBsAg), antibodies to HBV core antigen (HBc-Ab), and HBV-DNA in donated blood in Aseer Region (Southern part of Kingdom of Saudi Arabia) to detect prevention of the residual risk of transfusion transmitted HBV, donors in window period of HBV infection and the prevalence of HBV infection 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 hepatitis B surface antigen (HBsAg), anti-HBc, hepatitis C virus (HCV), human immunodeficiency virus (HIV) 1 and 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, HCV-RNA, and HIV-RNA by nucleic acid test (NAT) technology. Of 6698 (19 females (0.28%) and 6679 males (99.72)) blood donors screened, with median age of 28 (female) and 30 years (males), 69 (1.03%) were HBsAg positive of them 64 (0.96) were positive to HBV-DNA, 411 (6.14%) were anti-HBc positive of them 73 (1.09%) were positive to HBV-DNA. Cases positive to both HBsAg and HBc-Ab were 68 (1.02%) of them 63 (0.94%) were positive to HBV-DNA. All HBsAg and HBc-Ab positive cases were shown to be among male volunteers. In conclusion, prevalence of HBsAg in Asser region is very low. The rate of HBc-Ab in units of blood donation is relatively high. The presence of HBV-DNA in HBc-Ab positive donations makes it risky for use. Vaccination program against HBV decreased the rate of HBV transmission.