Abstract Between November 2006 and June 2007, HIV-1 reverse transcriptase (RT) and protease (PR) genes of 67 ARV-naive Cambodian patients were amplified and sequenced. At inclusion, the median age and duration of HIV infection were 28 and 1.1 years, respectively. The median CD4 and HIV-1 RNA were 611 cells/ml [IQR: 525-759] and 4.0 log(10) copies/ml [IQR: 3.4-4.6]. Among 67 HIV-1 strains, 95.5% were CRF 01_AE viruses (n = 64) whereas three clustered with subtype B. RT analysis indicated that only 1 patient out of 67, presenting K103N and M184V mutations, was resistant to NVP/EFV and 3TC/FTC. No primary resistance to protease inhibitors was detected in 59 amplified protease genes. The 1.49% (IC 95%: 0.04-8.04%) prevalence of transmitted drug-resistant strains in drug-naive patients was low in our study. Surveys of drug-resistant transmitted viruses should be regularly performed regarding the increasing access to HAART in Cambodia.