Sequence variation in the putative nonstructural region 5b (NS5b) of hepatitis C virus (HCV) was analyzed in China. Complementary DNA fragments from sera of 49 Chinese patients were amplified by polymerase chain reaction (PCR) and the products were cloned and sequenced. Based on the comparison in NS5b of 33 clones of genotype 1b and 16 clones of genotype 2a, Chinese isolates of HCV belong to the same subtype as HCV-J, and HC-J6 from Japan. There does exist, however, some heterogeneity in the primary structure of the nucleotide acid. Higher homology was found among Chinese isolates than among Chinese isolates and Japanese isolates. Furthermore, among Chinese isolates, we found some conserved nucleotide acid positions different from those of Japanese isolates. Comparison of average homology among the 33 clones of genotype 1b and the 16 clones of genotype 2a indicated that the average homology among genotype 2a was lower than that among genotype 1b. In addition, a deletion of three nucleotide acids and a frame-shift, resulting in the introduction of an in-frame stop codon, were first observed in the NS5b region. These results indicated geographical differences in the distribution of individual HCV isolates, and the existence of a local variant in the same subtype. Our findings also suggested the need for further study on the sequence of genotype 2a, to improve diagnosis and help to advance the development of a vaccine.