Background: Hepatitis B virus infection is a global public health problem, the diagnosis of which is based on detection of hepatitis B surface antigen in the blood. In spite of this, transmission of HBV infection has been documented from hepatitis B surface antigen negative blood donations. There is paucity of data on prevalence of HBV markers in prospective donors who are HBsAg negative in Nigeria. Objective: The objective of this study was to determine the prevalence of antiHBc, HBeAg, anti-HBe among apparently healthy blood donors who were negative for hepatitis B surface antigen and to establish whether there is a need for routine Hepatitis B core antibody screening of blood units. Materials and Methods: A cross-sectional study was carried out among blood donors at the blood bank of the University College Hospital, Ibadan between May and October, 2013. The serum samples of 490 participants who were negative for HBsAg using Monolisa Ultra HBsAg ELISA kits by BIORAD France were further tested for anti-HBc, anti-HBs, HBeAg and anti-HBe using ELISA kits by DIAPRO Diagnostic Bioprobes Milano, Italy. A structured questionnaire was used to assess the socio-demographic characteristics and presence of possible risk factors.