A baseline study has been conducted to determine the polymorphism of reverse transcriptase, protease, and envelope genes of HIV-1 isolates from 146 antiretroviral drug-naive Cambodian patients including 22 seroconverters and 124 pregnant women having been diagnosed HIV positive for less than 1 year. Amplification of at least one gene was successful for 144 isolates. All three genes were obtained for 136 isolates. Subtyping showed that CRF01_AE was predominant (130 cases). According to the ANRS September 2004 list, polymorphism substitutions (>50% versus the subtype B consensus) of CRF01_AE at drug resistance positions were observed only in protease: I13V (81%), E35D (87%), M36I (100%), R41K (96%), and H69K (100%). Two strains bore one major resistance mutation to PIs: M46I and N88D. Five other strains carried drug resistance mutations to RTIs: K70R (one strain), V75M (three strains), and K101E (one strain). Of the isolates 4.9% had drug resistance mutations to antiretroviral drugs.