Hepatitis C virus (HCV) infection remains common among hemodialysis patients and its occurrence is related mainly to nosocomial spread. Although dialysis patients with HCV infection respond well to interferon-based therapy, relapse is frequent. This study aimed at a selected group of hemodialysis patients infected with HCV infection undergoing interferon therapy who achieved end-of-treatment virological response but became HCV-RNA positive again 6 months after end-of-treatment. It was evaluated whether de novo HCV-RNA positivity in these non-sustained responders occurred due to lack of clearance of HCV after the initial response to interferon-alpha (relapse) or due to re-infection with a new strain (re-infection). Genotyping by Inno-LiPA and by phylogenetic tree analysis using partial HCV-NS5B sequences at two evaluation points: pre-treatment (T0) and 6 months after end-of-treatment (T18). Non-sustained responders (n = 15) carried subtypes 1a (8 patients), 1b (4 patients), 3a (2 patients), and 4a (1 patient) before treatment. Identical subtypes were detected in 10 patients at T18. Five patients changed genotypes at T18, suggesting nosocomial re-infection. This study emphasizes the importance of epidemiologic measures to control the re-exposure of hemodialysis patients treated previously for HCV infection.