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Hepatitis B is one of the major transfusion transmitted infections. It forms a serious health problem globally. Prevalence of Hepatitis B infection varies across the different geographical areas. Aim and objectives of this study were to know the sero-prevalence of Hepatitis B infection in voluntary blood donors at the Department of Immunohaematology& Blood Transfusion of MGM Kamothe Hospital. This is a retrospective study of the last three years from January 2012 to December 2014. All the blood units collected were screened for the Hepatitis B, HCV, HIV I & II, Syphilis and Malaria. ELISA was done to detect the Hepatitis B surface antigen in all the blood donors as a marker of infection. High sero-prevalence rate was observed among the male donors (1.12%) than in female blood donors (0.01%). Age-wise sero-prevalence was found to be more in 31 -40 years of age group (0.54%) and in this study majority of sero-positive donors were younger than 40 years. To reduce the prevalence of the post-transfusion hepatitis, stringent donor screening procedure, 100% voluntary blood donations are more effective, in addition to, better awareness among donors and re-intensification of prophylactic programs at public level to ensure the safe blood donation.