OBJECTIVES: To compare the results of conventional serological tests and molecular technology (NAT, Nucleic Acid Amplification Test), identify donors in the diagnostic window period, and determine the prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) among the samples of blood donors blocked by serological screening. METHODS: A retrospective cross-sectional study was carried out by analyzing blood donor information contained in the database of 20 blood centers in Paraná, from January 2018 to December 2019. RESULTS: A total of 1,496 blood bags were reactive for HBV, HCV, or HIV in serological and/or NAT tests. The 20th Regional Health (RH) Unit had the greatest number of unfit individuals with altered screening for the three infections, with a prevalence of 0.70%. The lowest number of blocked blood donors occurred in the 15th RH, with a prevalence of 0.08%. The highest prevalence of HBV occurred in the 8th RH, with a reagent serology of 0.34% and a positive NAT of 0.17%. For HCV, the prevalence for reagent serology was 0.28%, while that for NAT was 0.02%, which occurred in the 20th RH. For HIV and for NAT, the prevalence of blood donors with positive serology occurred in the 20th RH, at 0.25% and 0.04%, retrospectively. The 13th RH had the highest prevalence of HIV in relation to NAT, that is, conventional serology in concomitance with NAT technology, at 0.07%. During the 2-year period, only 1 reactive donor in the 9th was found for NAT (HBV), in a diagnostic window. CONCLUSION: In Paraná's blood centers, the inability to donate due to HBV, HCV, and HIV, occurred mainly in initial donors, men, those with >8 years of education, aged 16-45 years, married, and O positive. The most affected regions were located in the west and northwest of Paraná. Most of the results showed a discrepancy between the methodologies used.