Allele-specific PCR (ASPCR) is a highly sensitive, and reproducible method for the study of minor HIV-1 variants harboring resistance mutations and is significantly less labor-intensive and time-consuming than other techniques used for similar purposes. Furthermore, ASPCR has multiple applications in HIV research: it provides earlier and more sensitive detection of evolving resistance mutations, a more accurate assessment of transmitted drug-resistant mutants and a better evaluation of resistance selection after post-exposure or mother-to-child-transmission prophylaxis programs. This article outlines the principles of ASPCR and illustrates technical challenges in the design and application of ASPCR protocols by describing ASPCR assays developed for detecting resistance mutations in the protease (PR)- and reverse transcriptase (RT)-coding regions of pol and env. The assays achieved sensitivities of <1% for the D30N mutation in HIV-1 PR, M184V and I mutations in RT, and V38A in gp41. This method can be easily adapted to the quantitative detection of other mutations in HIV-1 or other viruses by introducing minor modifications to the methods described. In addition, ASPCR can be used to assess the dynamics of mutant populations in the viral quasispecies in response to changing selection pressures, allowing inferences on viral fitness in vivo through mathematical modeling.