Background: Hepatitis B is an infectious disease of worldwide importance and of great interest to transfusion medicine. The Amazon region has areas of high endemicity, outlining a worrying scenario for transfusion and epidemiological safety. Objective: To analyze the profiles of serological and molecular markers for HBV of blood donors from HEMOAM. Methods: Blood donors with different patterns of reactivity in serological and molecular screening for HBV were tested for viral load by the qPCR method at the reference center for liver diseases in the state of Amazonas. Results: A total of 230,591 donors were tested, with 3104 (1.34%) found reactive for HBV and 2790 (89.9%) found reactive for isolated anti-HBc. Viral load was not detected in 100% of donors reactive only to HBsAg, while 100% of donors with positive anti-HBc and positive HBsAg or HBV NAT demonstrated a detectable viral load. We also detected one case of occult hepatitis B (0.03%) only with reactive HBV NAT and five donors (0.2%) with positive anti-HBc and HBV NAT. Conclusions: With this result, the great importance of the anti-HBc test for the unsuitability of blood donors was verified, as well as the fundamental introduction of the HBV NAT test in screening for hepatitis B in Brazilian blood banks, as this was the only way to detect the viral infection burden in asymptomatic donors who previously would not be treated, which contributed to the maintenance of the endemicity of hepatitis B in the Brazilian Amazon.