The Hepatitis B virus (HBV) causes liver infection that can be life–threatening and often leads to chronic liver disease, liver cirrhosis and liver cancer. A cross sectional study was conducted to determine Hepatitis B virus distribution and associated risk factors among some high risk groups in Enugu Metropolis. A total of 518 subjects were sampled over a period of 2 years, from January 2013 to January 2015. The subjects consist of 233 blood donors, 164 pregnant women, 37 diabetic patients, 62 HIV patients and 22 sickle cell patients. Approximately 5 ml of blood was aseptically collected from each volunteer and analyzed for hepatitis B surface antigen (HBsAg). All HBV positive patients were analyzed for liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST). Patients co-infected with HIV were analyzed for CD4+ lymphocyte count. Out of the 518 samples, 45 (8.7%) were positive for HBsAg. The prevalence of HBV was highest among HIV patients 16 (25.8%), followed by blood donors 20 (8.6%), pregnant women 9 (5.5%) and 0% for both sickle cell and diabetic patients. Higher prevalence of HBsAg was found among males 25 (10.2%) than females 20 (7.3%). Out of the 45 HBsAg positive patients, 8 (17.8%) had high ALT levels, while 9 (20.0%) had high AST levels. Abnormal levels of transaminases (ALT and AST) were more in HIV patients (43.8%) than in pregnant women (22.2%) and blood donors (10%). All patients co-infected with HIV-HBV had CD4+ lymphocyte count of less than 1000 cells/µl even though they were on combination antiretroviral therapy. The risk factors of HBV in this study were multiple sex partners, age and unemployment (P=0.000), (P=0.045) and (P=0.014) respectively. There was high prevalence of HBV among HIV patients and blood donors and an intermediate prevalence among pregnant women in Enugu Metropolis. A vaccination programme targeting these risk groups is highly recommended.