Many drug-resistance mutations in HIV-1 reverse transcriptase fall within cytotoxic T lymphocytes (CTL) epitopes, but studies of the response to these epitopes in patients with virological failure are lacking. We therefore compared IFN-γ ELISPOT responses to the YV9 epitope (RT181-189) covering the lamivudine resistance mutation, M184V, in HLA-A2(+) antiretroviral treatment (ART)-naive patients (n = 19), to those found in HLA-A2(+) patients with virological failure (n = 15). Ten ART-naive patients had an ELISPOT response to the wild-type epitope that cross-reacted with the mutant epitope. Two patients with virological failure showed a specific response to the 184V mutant epitope. Responses against YV9 were strongly associated (p = 0.005) with the presence of a 177E mutation, and the same tendency was observed in an independent cohort of patients (n = 22). These results indicate that variants in flanking residues may influence CTL responses to conserved subdominant HIV-1 epitopes.