Hepatitis B virus (HBV) remains a leading cause of chronic hepatitis, maternal complications, and neonatal deaths in sub-Saharan Africa. Pregnant women serve as a major reservoir for the persistence and ongoing transmission of hepatitis B virus and HIV in a generalized heterosexual epidemic. The aim of this study is to assess the epidemiology of Hepatitis B infection among pregnant women in South West-Nigeria. This is a cross-sectional study of 353 pregnant women across 10 health facilities in the region. Results showed that of the 353 pregnant women tested, 37 were positive for the HBV antigen giving a prevalence estimate of 10.5% (95% CI: 7.5%-14.2%). We found significant negative association between odds of HBV infection and knowledge of HBV transmission through sex (OR: 0.30: 95%CI-0.11-0.82) and a positive association with blood transfusion in the past three months (OR: 9.5: 95% CI-1.58-57.14). Findings strongly suggest high endemicity of HBV and the possible implication of blood transfusion as a major route of ongoing HBV transmission among pregnant women in south-western Nigeria. We recommend further study of a prospective design to investigate the possible causal link between blood transfusion and the risk of HBV infection among pregnant women in Nigeria.