Introduction: Hepatitis C virus is the leading cause of chronic liver disease worldwide. The main route of transmission of HCV is a percutaneous exposure to infected blood. The risk of transmission is greatest after transfusion of blood and blood products from infected blood donors or repeated percutaneous exposure. The disadvantage of serological testing is that infection can not be proved during the window period. To shorten the time of the window period, and thus the risk of transmission of infection, molecular methods have been introduced for the detection of viral genomes in blood (HCV-RNA). Objectives: The aim of this study was to determine the number of true positive results using transcription mediated amplification (TMA) method and analyze the correlation of serological and molecular techniques for the detection of HCV infection. Material and methods: The survey instrument were the results of voluntary blood donors serological tests tested at the Department of serological and immunohaematological diagnostics of Clinical Institute of Transfusion Medicine, Clinical Hospital Centre Osijek and molecular tests (HCV-RNA) tested at the Department of NAT testing of blood donors at Croatian Institute for Transfusion Medicine. Results: From May 1st 2013 until May 1st 2016, total amount of blood donation was 81 225. From a total of 95 initially reactive results, four of them confirmed reactive for hepatitis C. Conclusion: Blood donors represent population with the lowest seroprevalence of HCV infection in the Republic of Croatia. The results of this study (0,005 %) correspond to the results compared to the results for the Croatian Republic and Western Europe (0 to 0.009 %).