Genotyping of hepatitis C virus (HCV) isolates is of particular interest for epidemiology. Because of extreme genome variability of HCV genome, each genotyping method should be evaluated in local setting before being introduced as a routine diagnostic procedure or a tool for large-scale epidemiological studies. This comparative study of four most widely used HCV genotyping assays, employed on 40 HCV isolates in Slovenia, showed that none of them was really suitable for genotyping Slovenian HCV isolates. Thanks to its satisfactory sensitivity, specificity and simplicity, the line probe assay was found to be the most appropriate tool for both routine and large-scale epidemiological studies. Using this assay the prevalence of HCV genotypes in Slovenia was studied on 226 individuals infected with HCV. Significant differences in HCV genotype distribution observed between epidemiological groups in Slovenia indicate a close relationship between individual HCV genotypes and certain routes of viral transmission.