We analyzed a hepatitis C virus (HCV) transmission case in the hemodialysis unit of a private clinic by sequencing two genome regions of virus isolates from a number of patients attending this unit and some external controls. The analysis of 337 nucleotides (nt) in the NS5B region did not provide enough resolution to ascertain which patients were actually involved in the outbreak and the potential source. Nevertheless, this region allowed the exclusion of several patients as putative sources of the transmission case based on their genotypes and phylogenetic relationships. On the other hand, the analysis of several 472-nt-long clone sequences per sample in a more rapidly evolving region of the HCV genome, coding for the envelope proteins and encompassing hypervariable region 1, allowed us to establish the existence of at least two independent transmission events involving two different source patients and three recipients. The direction of the transmissions was further corroborated by different measures of genetic variability within and among samples.