Genetic recombination plays a significant role in the survival and evolution of hepatitis C virus (HCV), but methodological limitations have hindered the exploration of genetic recombination. HCV serotypes were evaluated in 104 patients with chronic hepatitis C when they initially presented in hospitals. Subsequently, HCV genotypes were analyzed using primers for core gene and NS5B gene. Near-complete nucleotide sequences of eight HCV isolates from two suspected patients with 2b/1b recombinant HCV were analyzed by amplification of nine overlapping regions of HCV-specific oligonucleotide primers at different time points: (i) at the first admission; (ii) before and (iii) after interferon therapy; and (iv) after development of hepatocellular carcinoma. The nucleotide sequence of eight HCV isolates obtained was 9,321–9,471 nucleotides in length, comprising a single ORF (polyprotein of 3,014 amino acids.) and segregated into discordant genotypes of 2b and 1b HCV with a recombination junction in NS2. This study highlights the need for more precise characterization of HCV in clinical samples where there is a discrepancy between immunoassays and sequencing. It also demonstrates the circulation of novel inter-genotypic recombinant HCV in Japan, because the cross over point of 2b/1b recombinant HCV in eight clinical isolates of these two patients differed from previously reported HCV recombinant from the Philippines and Japan. J. Med. Virol. 84: 1018–1024, 2012. © 2012 Wiley Periodicals, Inc.