HIV subtype-specific data on mutation type, rate, and accumulation following HAART treatment failure are limited. We studied patterns and accrual of drug resistance mutations in a Cambodian CRF01_AE-infected cohort continuing a virologically failing first-line, nonnucleoside reverse transcriptase inhibitor- (NNRTI-) based, HAART. Between 2005 and 2007, 837 adult HIV-infected patients had regular plasma HIV-1 RNA viral load measurements at Sihanouk Hospital Centre of Hope (SHCH), Cambodia. Drug resistance testing was performed in all patients with HIV-1 RNA >1000 copies/ml after at least 6 months of HAART. Seventy-one patients with a mean age of 34 years, of whom 68% were male, were retrospectively assessed at virological failure. The median duration of antiretroviral therapy was 12.3 (IQR 7.1-18.23) months, the median CD4 cell count was 173 (IQR 118-256) cells/mm(3), and the mean plasma HIV-1 RNA viral load was 3.9 log (SD 0.72) at failure. NNRTI mutations, M184I/V mutation, thymidine analogue mutations, and K65R were observed in 78.9%, 69%, 20%, and 12.7% of patients, respectively. For 33 patients, genotypic testing was carried out on at least two occasions before the switch to second-line HAART after a median duration of 5.8 (IQR 4.3-6.1) months of virological failure: 54.5% of patients accumulated new mutations with a rate of 1.6 mutations per person-year. Accumulation was seen both for nucleoside and nonnucleoside reverse transcriptase inhibitors, and also in patients with low-level viremia. Subtype-specific data on mutation type, rate, and accumulation after HAART failure are urgently needed to optimize treatment strategies in resource-limited settings.