In a large cohort in rural South Africa, 73% of subtype-C-infected patients initiating highly active antiretroviral therapy achieved viral suppression. In patients with subsequent virological failure, an unexpected, rapid accumulation of nonnucleoside reverse transcriptase inhibitor-associated mutations was observed, whereas no thymidine analogue-associated mutations emerged. It appeared that several patients had drug-associated mutations prior to starting antiretrovirals, suggesting that transmission of resistance may have contributed to the accumulation of nonnucleoside reverse transcriptase inhibitor-mutations. Importantly, monitoring of HIV-RNA and prompt switch of treatment may prevent development of thymidine analogue-associated mutations.