As the number of HIV-1-infected individuals receiving antiretroviral drugs has been rapidly increasing in developing countries, there is an urgent need for drug resistance genotype information of non-B subtype HIV-1 and for the establishment of a practical system of monitoring drug-resistant viruses. This study first sequenced the reverse transcriptase region of HIV-1 in 112 infected individuals who had been treated with zidovudine (AZT)/didanosine or AZT/zalcitabine as dual therapy at a government hospital in northern Thailand and then compared the above sequence method with mutagenically separated polymerase chain reaction (MS-PCR) for detecting M41L and K70R mutations. Concordant rates of detecting M41L and K70R mutations by the 2 methods were 96.9% (93/96) and 92.7% (89/96), respectively. The M41L and K70R MS-PCR could detect 86.4% of AZT-resistant strains with any resistance mutation, which was determined by the sequencing method. Then 292 drug-naive individuals were screened for the presence of drug-resistant HIV-1 by the MS-PCR assay and it was found that 2 individuals (0.7%) carried viruses with either the M41L or K70R mutation. It is feasible to test a large number of samples with MS-PCR, which is sensitive, cheap, and easy to perform and does not require sophisticated equipment. The M41L and K70R MS-PCR is potentially a useful tool to monitor the spread of AZT-resistant HIV-1 in resource-limited countries.