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Although the risk of transfusion-transmitted hepatitis B virus (TT-HBV) infection is very low, it still exists. Therefore, introduction of further precautions to reduce this risk is discussed at present. However, so far no data are available about the HBV vaccination status among blood donors (BDs). We compared HBV vaccination status of apheresis donors (ADs) of a university based and whole BDs (WBDs) of a Red Cross blood donation service using a standardized questionnaire. On the basis of these data, the estimated costs over 10 years for HBV vaccination were calculated for two different strategies and compared with the costs for HBV nucleic acid amplification technology (NAT) testing. 22.3% of the WBDs and 41.2% of the ADs indicated having received at least one HBV vaccine dose. This difference was related to the different demographic structures of the two BD populations (BDPs). With regard to the primary costs for the blood donation service, HBV vaccination of BDs could be an alternative to HBV NAT testing, especially for BDPs with an already high HBV vaccination rate and a high donation frequency.