The purpose of our study was to examine the emergence of the Y181C resistance mutation in an NNRTI-naive subject (index patient) at different time points. Phylogenetic trees in protease (PR) and partial reverse transcriptase (RT) regions were inferred by the maximum likelihood (ML) method. The Y181C mutation was detected for the first time when the patient was receiving d4T + ddI + LPV/r; the previous drug combination was 3TC + AZT + IDV. The particular mutation (Y181C) was not present at any time point during the treatment period with 3TC + AZT + IDV. Moreover, there was no evidence of resistance mutations in RT before the initiation of antiretroviral therapy. Phylogenetic analysis including sequences from the index patient and his spouse sampled at different time points, as well as control sequences belonging to the same HIV-1 subtype, revealed that there is no evidence of coinfection or reinfection with Y181C resistance strains, while the virus for both subjects was classified as subtype CRF14_BG. Overall, our findings suggest that the Y181C resistance mutation may be selected, not only by NNRTIs, but also by d4T. This may be of particular significance in developing countries where treatment with Triomune, a fixed combination of d4T, ddI, and nevirapine, is common. The genetic barrier against resistance of this combination may be lower than previously thought.