Blood donor screening of viral markers in Poland is based on serologic testing for anti-HCV, HBsAg, anti-HIV1/2 (chemiluminescence tests) and on nucleic acid testing (NAT) for RNA HCV, RNA HIV-1 and DNA HBV performed in minipools of 6 with real-time PCR (MPX 2.0 test on cobas s201) or with TMA in individual donations (Ultrio Plus or Ultrio Elite). Donors of plasma for anti-D and anti-HBs production are tested for parvovirus B19 DNA. Before implementation tests and equipment are evaluated at the Institute of Hematology and Transfusion Medicine (IHTM). The last 20 years witnessed a decreasing trend for HBsAg in both first time and repeat donors (1%-0.3% and 0.1%-0.02% respectively). Prevalence of anti-HCV repeat reactive results was stable and oscillated around 0.8% for first time donors and 0.2% for repeat donors. Elevated prevalence of seropositive HIV infected donors was recently observed (7.5-9 cases/100,000 donors). Since respective molecular markers implementation HCV RNA was detected on average in 1/119,235 seronegative donations, HIV RNA in 1/783,821 and HBV DNA in 1/61,047. HBV NAT yields were mostly occult hepatitis B (1/80,248); window period cases were less frequent (1/255,146). The efficiency of HBV DNA detection depends on the sensitivity of the HBV DNA screening system.