We analysed hepatitis C virus (HCV) sequences to determine whether nosocomial transmission of HCV occurred in a haemodialysis unit. Twenty patients positive for serum HCV RNA were investigated. All were undergoing haemodialysis therapy in the same room. The hypervariable region 1 (HVR1) sequence of HCV was amplified and multiple clones sequenced. Phylogenetic analysis of these sequences revealed five genetic clusters consisting of HCV isolates from 11 of the 20 patients. In addition to two genetic clusters of HCV isolates from the four currently seroconverting patients and another patient who had been persistently infected, we identified three other phylogenetic relationships in HCV isolates from six patients. The patients grouped into the same cluster received haemodialysis individually on the same shift and/or side-by-side. Phylogenetic analysis of HCV HVR1 sequences corroborated the patient-to-patient HCV transmission suggested by an epidemiological study and that unrecognized transmission of HCV occurs in the dialysis room. Our multiple clone analysis of HCV isolates provides detailed information on nosocomial transmission of HCV. Transmission occurs more frequently when treatment is performed at the same time than in consoles located close to each other.