The genotypic distribution of hepatitis C virus (HCV) isolated from blood donors from four major regions of Thailand was studied by reverse hybridization assays. PCR-amplified products from the 5 noncoding and core regions of the viral genome were hybridized to genotype-and subtype-specific probes which were immobilized on the nitrocellulose membrane. Of 332 anti-HCV-positive plasma samples studied, 71% contained HCV RNA. HCV genotype 3a was the most prevalent genotype (39%), followed by genotype 1b (20%) and genotype 6 group variants (18%). HCV genotype 1a was identified among 9% of all isolates. Other genotypes (genotype 1 which was neither 1a nor 1b, genotype 3b, and an unclassified genotype) were uncommon. There was no difference in the mean age of the donors infected with different HCV genotypes. The genotypic distribution pattern of HCV was similar among HCV isolates from different regions of Thailand.