Background: This cross-sectional study investigated the serological profile, socio-demographic characters and risk factors of hepatitis B virus (HBV) infection among HBsAg-negative blood donors at the University of Abuja Teaching Hospital (UATH) Gwagwalada and Nnamdi Azikiwe University Teaching Hospital (NAUTH) Nnewi, Nigeria.Methods: Blood samples collected from 176 HBsAg-negative blood donors (96 from UATH and 80 from NAUTH) were screened using a commercially available HBV 5-Panel (CTK Biotech, USA) and anti-HBc IgM ELISA kits. Determination of HBV-DNA  was done on 36 HBV positive and 100 negative samples using COBAS Roche Real-time qPCR. Structured questionnaires were used to collate subjects' socio-demographic variables and risk factors of HBV infection. Results: Out of 176 samples tested, 140 (79.5%) were negative for HBV serologic markers while 36 (20.5%) were positive. The pattern of seropositivity showed that 19 (10.8%) samples were positive for anti-HBs, 9 (5.1%) were positive for anti-HBc, 3 (1.7%) were positive for both anti-HBc & anti-HBs and 5 (2.8%) were positive for anti-HBc IgM. None was positive for HBeAg and anti-HBe markers. Of the 36 HBV positive and 100 negative samples, 15 (41.7%) and 3 (3%) were positive for HBV-DNA respectively p=0.006. Not heard of HBV, no vaccination with HBV vaccines, previous blood transfusion, history of sexually transmitted diseases and visiting commercial barbers were significantly associated with HBV infection. Socio-demographic data showed that male blood donors were more infected with HBV than the females (p=0.284) and age group 26-40 years old was more affected than other age groups (p=0.015). Conclusions: The study revealed the endemicity of HBV infection and recommends that blood donors with critical risk factors be deferred from blood donation to reduce HBV transmission risk in Nigeria. Anti-HBc and anti-HBs markers could be included as screening tests for blood donors since HBV-DNA testing is not readily available and affordable.