A novel assay is described for the detection of HIV-1 drug resistance that is simple, cheap and sensitive. HIV-1 drug resistance in B and non-B HIV-1 subtypes was investigated using Mutagenically-Separated PCR (MS–PCR) — a competitive semi-nested PCR which uses mutagenic primers. The assay was assessed for sensitivity, specificity and its ability to detect mutant virus within a mixed mutant–wild-type population. Gene sequencing was carried out simultaneously for comparison. MS–PCR detected five copies of HIV-1 RNA from laboratory isolates and 50 copies from patient samples. We demonstrate 100% specificity of detection for wild type or mutant virus for clades A, B, C, D and E. For mixed populations of virus, MS–PCR can detect at least a 10% mix of wild type:mutant, or vice-versa. When applied to African patient samples MS–PCR detected 91.6% of the codons tested. Concordance with sequencing data was 88.8% for protease and 97.2% for RT. MS-PCR is sensitive and specific for the detection of mutations in HIV-1, and can be adapted easily to test for resistance at any codon of interest.