Hepatitis C virus (HCV) variation in specific T-cell epitopes may represent a mechanism of viral persistence in chronic infection. We examined the HCV non-structural protein 3 (NS3), including the immunologically relevant epitopes HCV NS3-2 KLVALGINAV (human leukocyte antigen [HLA]-A2-restricted) and HCV NS3-1391 LIFCHSKKK (HLA-A3-restricted), in 22 HLA-A2+ patients with chronic infection. Significant amino acid variation was found in HCV NS3-2 epitope sequences when compared to the HCV-1 prototype virus. Six of the nine different HCV NS3-2 peptide variants were identified in patients with HCV NS3-2-specific CD8+ cells, detected with an HLA-A2 tetramer made with the HCV-1 prototype peptide. Phylogenetic analysis, including HCV reference sequences other than HCV-1, suggested however that most of the variations in the HCV NS3-2 epitope could be related to genetic heterogeneity between HCV reference subtypes. Variation was less common when comparing HCV NS3-2 epitope sequences from the clinical isolates to the most-closely related HCV reference subtype in each case. Some subtype-independent variations were found in epitopic residues probably important for T-cell receptor interaction. In contrast, no significant variation was found in HLA primary anchor sites, flanking regions, or in the contiguous HLA A3-restricted CD8+ T-cell epitope. Ongoing variation was not evident in two selected patients with follow-up. In conclusion, (i) the HCV NS3-2 epitope is not conserved between different HCV strains/subtypes, and (ii) an HLA-A2 tetramer loaded with the HCV-1 prototype NS3-2 peptide may still detect NS3-specific CD8+ cells in some patients with variant viruses. These data may be useful to improve T-cell assays using HCV NS3 peptides, taking into account the genetic diversity of this virus.