We investigated the unrecognized patient-to-patient transmission of hepatitis C virus (HCV) in hemodialysis units by performing phylogenetic and serological analyses of hypervariable region 1 (HVR1) of HCV. Of the 62 patients in one center, 11 were positive for HCV RNA. A total of 24 HVR1 sequences, including the minor population of sequences of HCV isolates, from each patient were closely related and classified into five clusters by phylogenetic analysis. Of the 11 patients, 5 were infected with multiple clusters of HCV. Two patients were infected with HCV during an 18-month interval between examinations, and these HVR1 sequences fell into one of the five clusters. In another hemodialysis center, 5 of the 20 patients were HCV RNA positive, and two HVR1 sequences were found to be closely related and phylogenetically derived from the same cluster. The antibody responses of these patients to the HVR1 peptides representative of the genetic clusters revealed exactly the same clustering as that shown by phylogenetic analysis. These findings suggest that phylogenetic and serological analyses of HVR1 sensitively detect unrecognized and multiple transmission of HCV occurring within the same room in hemodialysis centers. Fingerprinting analyses using hypervariable regions of infectious agents are useful in identifying the precise route of transmission of infection.