Background:Although sensitive screening assays for hepatitis B virus surface antigen (HBsAg) are available, special cases of post-transfusion hepatitis B virus infection still occur. The present study was conducted to evaluate the prevalence of anti-hepatitis B core (anti-HBc) positivity and the presence of HBV-DNA in serum samples of healthy blood donors negative for both HBsAg and anti-HCV antibodies in Benha, Egypt. Materials and methods: The study included a screening of 450 selected blood donors. The distribution of blood donors was 288 males (64%) and 162 females (36%). The recruited blood donors who met the criteria for blood donation were routinely screened for HBsAg, HIV I/2-Ab, and syphilis-antibodies. The blood units for donations were further analyzed for the presence of HBc-IgM and HBV-DNA levels by PCR method. Results: Testing of the accepted units for the donation was about 12 (2.7%) HBc-IgM positive, and 9 (2%) HBV-DNA positive units. The standard screening of blood unit failed to recognize early acute or window HBV infection where HBsAg is missing. Conclusions:Our investigations proposed that sensitive methods for the detection of HBV by PCR might be recommended in the screening of donated blood. Furthermore, anti-HBc antibodies should be tested regularly on all blood donation units.