Hepatocellular carcinoma (HCC) is one of the common sequelae of hepatitis C virus (HCV) infection. It remains controversial, however, whether HCV itself plays a direct role in the development of HCC. Although HCV core, NS3, and NS5A proteins were reported to display tumorigenic activities in cell culture and experimental animal systems, their clinical impact on HCC development in humans is still unclear. In this study we investigated sequence polymorphisms in the core protein, NS3, and NS5A of HCV genotype 1b (HCV-1b) in 49 patients who later developed HCC during a follow-up of an average of 6.5 years and in 100 patients who did not develop HCC after a 15-year follow-up. Sequence analysis revealed that Gln at position 70 of the core protein (core-Gln70), Tyr at position 1082 plus Gln at 1112 of NS3 (NS3-Tyr1082/Gln1112), and six or more mutations in the interferon/ribavirin resistance-determining region of NS5A (NS5A-IRRDR≥6) were significantly associated with development of HCC. Multivariate analysis identified core-Gln70, NS3-Tyr1082/Gln1112, and α-fetoprotein (AFP) levels (>20 ng/L) as independent factors associated with HCC. Kaplan-Meier analysis revealed a higher cumulative incidence of HCC for patients infected with HCV isolates with core-Gln70, NS3-Tyr1082/Gln1112 or both than for those with non-(Gln70 plus NS3-Tyr1082/Gln1112). In most cases, neither the residues at position 70 of the core protein nor positions 1082 and 1112 of the NS3 protein changed during the observation period. Conclusion: HCV isolates with core-Gln70 and/or NS3-Tyr1082/Gln1112 are more closely associated with HCC development compared to those with non-(Gln70 plus NS3-Tyr1082/Gln1112). (HEPATOLOGY 2013;58:555-563)