An epidemiological and molecular study of hepatitis C virus (HCV) infection was carried out in Brazilian hemodialysis centers. A total of 1,095 patients in all 15 hemodialysis centers in the State of Goiás, Brazil, were studied. All patients were interviewed for possible risk factors to HCV infection and serum samples tested for anti-HCV by ELISA and for HCV RNA by nested RT-PCR of the 5' NC region. For sequence analysis, HCV RNA amplification for the NS5B region (nt 8,279-8,619) was performed. The phylogenetic tree was generated with MrBayes, and clusters with support values above 0.85 were considered epidemiologically related. Of the 1,095 patients, 180 were anti-HCV and/or HCV RNA positive, resulting in an overall prevalence of 16.4% (95% CI: 14.3-18.7). The prevalence of HCV infection in the dialysis centers ranged from 0% to 47.7%. Multivariate analysis of risk factors revealed that history of blood transfusion not screened for anti-HCV and length of time on hemodialysis were independently associated with HCV infection in this population. One hundred six samples could be amplified and sequenced in the NS5B region. Among them, plylogenetic tree analysis revealed that 69 sequences form 13 separated clusters, which were supported by credibility intervals ranging from 85% to 100%, indicating a very close relationship among the HCV isolates and therefore a likely transmission of the virus between patients. By combining phylogenetic analysis with epidemiological data, routes of transmission between the clustered-related-patients could be suggested. These findings provide evidence for nosocomial transmission of HCV in Brazilian hemodialysis centers.