CRF01_AE is a major subtype of human immunodeficiency virus type 1 (HIV-1) circulating in Southeast Asia, including Thailand. We performed genotypic studies on HIV-1 CRF01_AE protease (PR) and reverse transcriptase (RT) derived from plasma samples from drug treatment-naive patients residing in central Thailand. Direct sequencing of amplified CRF01_AE PR and RT genes revealed that drug resistance-associated as well as background mutations were frequently detected in CRF01_AE PR. In contrast, although several background mutations were detected, no drug resistance-associated mutations were observed in CRF01_AE RT. Antiretroviral drugs, including PR inhibitors, have been increasingly used in Thailand. To achieve effective antiretroviral therapy, we propose that it is important to reveal the prevalence of drug resistance-associated mutations among drug treatment-naive patients by further surveillance studies.