Despite the decrease in transfusion-transmitted hepatitis B virus (HBV), it is still one of the main problems in transfusion medicine. Recent studies have shown that HBV is transmissible to recipients from HBsAg negative donations. This study was aimed to detect anti-HBc, anti-HBs and HBV-DNA positivity in HBsAg negative cases and to determine whether a reconsideration is required for HBV screening policies in blood banking. In this study, anti-HBs and anti-HBc total was investigated in 9282 HBsAg negative donor samples by commercial ELISA (Orto-Clinical Diagnostics, Vitros, Brazil) system and HBV-DNA by real-time PCR method (QIAGEN, Artus 3000, Germany). In 9282 HBsAg negative samples, anti-HBc positivity rate was 18% (1679/9282). Anti-HBs negativity was detected in 15% (n=225) of these anti-HBc positive sera. Among the isolated anti-HBc positive (HBsAg negative, anti-HBc positive, anti-HBs negative) 225 serum samples 218 were investigated for HBV-DNA presence and one sample was found to be positive (0.45%). When the samples not tested due to insufficient serum amount were excluded, the total rate of isolated anti-HBc positivity in the study region was 2.5% (225/9107) and HBV-DNA positivity in the HBsAg negative group was 0.011% (1/9100). The annual number of blood donations in our center was about 21.000, thus the rate of HBV transmission from HBsAg negative donors could be estimated as 2.3 patients/year. Although the rate of HBV transmission due to HBV-DNA positivity among HBsAg negative blood donors is low, this risk should always be kept in mind in transfusion medicine.