The hepatitis C virus (HCV) quasispecies nature in the hypervariable region (HVR) has been reported and found to relate to the effectiveness of interferon (IFN) treatment. However, the quasispecies nature in the nonstructural (NS) 5b region remains to be addressed. To examine this characterization and relationship with IFN therapy, we sequenced six independent HCV clones from each of eight patients. The eight patients were classified as responders or nonresponders to IFN. In the four responders, we found one to three isolates in each of the six clones. In the nonresponders, the six clones consisted of four, five, six, and six isolates, respectively. Compared the (NS) 5b genes of the isolates obtained from the patients with that of the reported hepatitis C virus HC-C2 or HC-J6 isolate the ratio of nonsynonymous to total substitutions ranged from 17.61% to 30.95% in the responders and from 33.11% to 76.47% in the nonresponders. We also compared posttreatment with pretreatment sequences. The average number of varying amino acids ranged from 5.5 to 9.0 in isolates remaining after IFN treatment and from 4.3 to 5.5 in the isolates that disappeared with IFN treatment. Two changed amino acids (glycine to arginine and valine to isoleucine) (compared with the pretreatment clones) were found in the posttreatment clones of one of the responders and one amino acid change (valine to alanine) was found in another responder. These results suggest that the NS5b quasispecies correlates with IFN treatment effectiveness. These results also implied that the heterogeneity in different hierarchical strata has a common impact on IFN treatment, making infected patients resistant to IFN. Our study also provides evidence that HCV elimination and mutation may occur simultaneously during IFN therapy.