HIV-infected children are at high risk of acquiring drug-resistant viruses, which is of particular concern in settings where antiretroviral drug options are limited. We aimed to assess resistance patterns and predict viral drug susceptibility among children with first-line antiretroviral therapy (ART) failure in Uganda. A cross-sectional analysis of children switching ART regimens due to first-line failure was performed at three clinical sites in Uganda. HIV-RNA determination and genotypic resistance testing on all specimens with HIV-RNA >1000 copies/ml was performed. Major drug resistance mutations were scored using the 2011 International Antiviral Society-USA list. The Stanford algorithm was used to predict drug susceptibility. Drug classess At the time of switch, 44 genotypic resistance tests were available for 50 children. All children harbored virus with non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance (95% confidence interval [CI] 92-100%) and NRTI resistance was present in 98% (95% CI 88-100%). Forty-six percent (95% CI 30-61%) of children harbored ≥ 2 thymidine analogue mutations. M184V was identified as the only NRTI mutation in 27% (95% CI 15-43%). HIV susceptibility to NRTIs, with the exception of tenofovir, was reduced in ≥60% of children. Ugandan children experiencing first-line ART failure in our study harbored high rates of dual-class and accumulated HIV drug resistance. Methods to prevent treatment failure, including adequate pediatric formulations and alternative second-line treatment options are urgently needed.