INTRODUCTION: Hepatitis B viral infection represents a major public health problem. In order to reduce the transmission of hepatitis B through blood transfusion and because of the inability to detect occult hepatitis B viral infection by serological methods, molecular ID-NAT testing was introduced. Furthermore, the HBV DNA is directly identified. OBJECTIVES: The aim was to determine the number of positive donor samples after confirmatory serological and molecular diagnosis of HBV. By comparison of serological and molecular diagnostics category of HBV infections all of the positive donor patterns as well as the number of permanently suspended blood donors have been identified. Furthermore, by determining the number of permanently suspended donors the reduction of possible HBV through blood transfusion was calculated. MATERIALS AND METHODS: The data comprised of the results of the compulsory serological screening of HBV blood donors at the Clinical Institute for Transfusion Medicine at Clinical Hospital Centre Osijek, the results of validated tests, and the results of molecular ID-NAT and discriminatory HBV (dHBV) tests obtained at the Croatian Institute for Transfusion Medicine, Zagreb. RESULTS: After the analysis, various forms of HBV in blood donors were identified: chronic and occult hepatitis B virus infection and previous contact with HBV. Acute HBV infection as well as infection during the diagnostic window was not observed. The correlation between serological and molecular diagnosis has been demonstrated in cases of chronic HBV infections. There were 26 blood donors, 25 men and one woman permanently suspended from donating blood. 76 blood preparations produced from donations of 26 permanently delayed blood donors were destroyed and thus their use was directly prevented. CONCLUSION: The actual number of HBsAg serologically reactive results in percentage is 0.011% less than the prevalence of HBsAg in the Republic of Croatia, which is more than 2%. In comparison with serum test results at the Croatian Institute for Transfusion Medicine for 2013, 2014 and 2015, the prevalence rate is somewhat higher (0.011 %), since the percentage of serologically positive results for that year was 0.008 %, 0.008 % and 0.006.