The establishment of clonal infection of hepatitis C virus (HCV) in a small-animal model is important for the analysis of HCV virology. A previous study developed models of molecularly cloned genotype 1a and 2a HCV infection using human hepatocyte-transplanted chimeric mice. This study developed a new model of molecularly cloned genotype 1b HCV infection. A full-length genotype 1b HCV genome, HCV-KT9, was cloned from a serum sample from a patient with severe acute hepatitis. The chimeric mice were inoculated intrahepatically with in vitro-transcribed HCV-KT9 RNA. Inoculated mice developed viraemia at 2 weeks post-infection, and this persisted for more than 6 weeks. Passage experiments indicated that the sera of these mice contained infectious HCV. Interestingly, a similar clone, HCV-KT1, in which the poly(U/UC) tract was 29 nt shorter than in HCV-KT9, showed poorer in vivo infectivity and replication ability. An in vitro study showed that no virus was produced in the culture medium from HCV-KT9-transfected cells. In conclusion, this study developed a genetically engineered genotype 1b HCV-infected mouse. This mouse model will be useful for the study of HCV virology, particularly the mechanism underlying the variable resistance of HCV genotypes to interferon therapy.