Drug-resistant viruses may be present as minority variants during early treatment failures or following discontinuation of failed antiretroviral regimens. A limitation of the traditional direct PCR population sequencing method is its inability to detect human immunodeficiency virus type 1 (HIV-1) variants present at frequencies lower than 20%. A drug resistance genotyping assay based on the isolation and DNA sequencing of minority HIV protease variants is presented here. A multiple-codon-specific heteroduplex generator probe was constructed to improve the separation of HIV protease genes varying in sequence at 12 codons associated with resistance to protease inhibitors. Using an RNA molecule as probe allowed the simple sequencing of protease variants isolated as RNA/DNA heteroduplexes with different electrophoretic mobilities. The protease gene RNA heteroduplex generator-tracking assay (RNA-HTA) was tested on plasma quasispecies from 21 HIV-1-infected persons in whom one or more protease resistance mutations emerged during therapy or following initiation of salvage regimens. In 11 of 21 cases, RNA-HTA testing of virus from the first episode of virologic failure identified protease resistance mutations not seen by population-based PCR sequencing. In 8 of these 11 cases, all of the low-frequency drug resistance mutations detected exclusively by RNA-HTA during the first episode became detectable by population-based PCR sequencing at the later time point. Distinct sets of protease mutations could be linked on different genomes in patients with high-frequency protease gene lineages. The enhanced detection of minority drug resistance variants using a sequencing-based assay may improve the efficacy of genotype-assisted salvage therapies.