The use of structured treatment interruption (STI) in human immunodeficiency virus (HIV)-infected subjects is currently being studied as an alternative therapeutic strategy for HIV-1. The potential risk for selection of drug-resistant HIV-1 variants during STI is unknown and remains a concern. Therefore, the emergence of drug resistance in sequential plasma samples obtained from 28 subjects with chronic HIV infection was studied. They underwent 4 cycles of 2-week STI, followed by 8-week retreatment with highly active antiretroviral therapy identical to that used before STI, and they had never failed treatment before undergoing STI. At week 40, treatment was stopped for a longer period. Minor populations of drug-resistant variants were detected by quantitative real-time polymerase chain reaction, by use of allele-discriminating oligonucleotides for 2 key resistance mutations: L90M (protease) and M184V (reverse transcriptase). The approximate discriminative power was 0.1%. In 14 of 25 and in 3 of 25 subjects, the M184V and the L90M mutations, respectively, were detected as minor populations, at different times during STI. Overall, these results indicate that, in subjects undergoing multiple STIs, HIV-1 variants carrying drug-resistance mutations can emerge during periods of increased HIV-1 replication.