Background: Three patients attending a hemodialysis unit were diagnosed with acute hepatitis C virus (HCV) infection. We investigated the scope and mode of transmission. Methods: Patients and staff were tested to determine HCV infection status; all HCV-RNA-positive sera underwent quasispecies analysis to assess genetic relatedness. Staff practices were evaluated via interviews and observations. A cohort study was performed to assess risk factors for incident HCV infection. Results: HCV infection was documented at time of hire or unit admission for 2 staff and 12 patients (prevalent case-patients). Seven (13%) of 52 patients HCV susceptible at admission to the unit subsequently acquired HCV infection (incident case-patients). Analysis of HCV quasispecies from the hyper variable region 1 identified 2 separate clusters each containing 3 incident case-patients and 1 prevalent case-patient. Incident case-patients received a higher median number of intravenous medications per dialysis session compared to susceptible patients (2.1 vs 1.8, p-value = 0.0606). Only one incident case-patient received dialysis on the same machine as their genetically related prevalent case-patient. Preparation of injection medications at the dialysis station on a mobile medication cart, and failures to clean environmental surfaces between patients were infection control breaches identified as likely modes of HCV transmission. Conclusions: Epidemiologic and laboratory data revealed transmission of HCV among patients at the same dialysis unit. Transmission was most likely related to infection control breaches. Our findings reinforce the risk of patient-to-patient HCV transmission in hemodialysis units when staff fails to adhere to recommended infection control practices.