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Hepatitis C virus (HCV) transmission is related to blood transfusion and transmission in hemodialysis (HD) units. The aim of the study was to investigate the distribution of HCV genotypes and routes of HCV transmission in three groups of anti-HCV positive patients in north-west Croatia. A total of 111 patients were studied. Patients were classified into three groups: 45 HD patients with a low percentage of anti-HCV positivity (group 1), 60 HD patients treated at another HD unit and with high percentage of anti-HCV positivity (group 2), and six anti-HCV positive patients with chronic hepatitis (group 3). Most of the HD patients were treated during the sama shift, but with separate equipment. Serum HCV RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Genotyping of HCV isolates was performed with a line-probe assay. Patients of groups 1 and 2 did not show significant differences with regard to the clinical profile. In group 1, all anti-HCV positive patients were RT-PCR positive and all of them were infected with the same genotype (genotype 3). In group 2, 89% of anti-HCV positive patients were RT-PCR positive and all infected with the same genotype (genotype 1 b). In group 3, 50% anti-HCV positive chronic hepatitis patients were RT-PCR positive. Two of them were infected with genotype 1 b and one with genotype 4. The homogeneity of HCV genotypes in the patients from both HD units (groups 1 and 2), seemed to indicate nosocomial transmission of HCV, whereas viremia was found to be related to blood transfusion in all group 3 patients. The exact mechanism involved in the transmission of HCV in HD units remains to be discovered.