In order to evaluate the usefulness of resistance testing after a viral rebound with plasma HIV RNA levels of less than 1,000 copies (c)/ml, genotyping was performed on 39 samples from patients on highly active antiretroviral therapy (HAART) showing a viremia of over 50 c/ml up to a maximum of 1,000 c/ml after at least one undetectable viral load result. Protease and reverse transcriptase (RT) sequences were obtained for all 39 samples. In 10 (25.6%) of the samples, mutations not seen before the initiation of the regimen were observed. The M184V/I mutation was the most prevalent but in several patients a combination of multiple mutations was detected. Follow-up samples were available for 34 patients. In six (85.71%) out of seven patients with new mutations, the viral load on the follow-up visit remained detectable, indicating true failure, compared to 6 (31.6%) true failures out of 19 patients in whom only wild type virus was detected (P = 0.02) and three (37.5%) out of eight patients in whom only the mutations already present at the initiation of HAART were seen (P = 0.08). The results indicate that reliable resistance testing can be performed on samples with a viral burden of less than 1,000 c/ml and demonstrate that multiple drug resistance mutations can be selected at low viral load rebounds. Most importantly, detection of resistance mutations in viral rebound samples was predictive of subsequent virological failure.