In HIV-1 subtype C infected populations in south India, we searched for novel mutations associated with failing antiretroviral therapy that included nucleoside reverse transcriptase (RT) inhibitors. HIV-1 RT sequences were generated from treated and untreated groups and each nucleotide position was analysed with appropriate corrections for multiple testing. We found that nonsynonymous mutations at positions 208 and 228 were strongly associated with the presence of thymidine analogue mutations in the treated group, and were not present at all in the naïve group. The role of these substitutions on treatment outcomes and the evolution of drug resistance in HIV-1 subtype-C infected populations warrant further investigation.