Introduction: Hepatitis B viral infection has been transmitted from donors with HBV infections who have negative HBs Ag. Many countries have implemented nucleic acid testing (NAT) to screen donors with non- reactive HBs Ag for detection of HBV DNA and enhance safety of blood transfusion, while it is restricted to limited blood banks in Egypt. The aim of our reserch to evaluate the significance of NAT technology in detection of HBV DNA in the Egyptian blood donors with HBs Ag non- reactivity. The study included 36,584 collected blood samples from volunteer blood donors at the blood bank of University Hospitals. Each specimen was tested for HBs Ag; non- reactive sera were further tested for qualitative detection of HBV-DNA by NAT testing. All positive HBV-DNA donors were tested for anti- HBc and anti- HBs by electro-chemiluminescence immunoassay and confirmed by quantitative RT-PCR. Among 34,671 donors non-reactive to HBs Ag, 34,657 (99.96%) were tested negative for HBV- DNA and 14 specimens (0.04%) were positive for HBV via NAT testing. Among HBV NAT positive donors, HBs Ab reactive only in (2); HBc Ab reactive only in (3); HBs and HBc Abs reactive in (3) while HBs and HBc Ab non-reactive in (6). All tested sera 14 (100%) showed low viral load for HBV (<50 IU/ml) confirmed by RT- qPCR. Our results highlighted the significance of the HBV NAT technique to reduce the potential risk of HBV transfusion-transmission and the critical need to enforce the usage of NAT technology in all blood banks in Egypt.