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Background Better understanding of drug resistance patterns in HIV-infected children on antiretroviral therapy (ART) is required to inform public health policies in high prevalence settings. The aim of this study was to characterise the acquired drug resistance in HIV-infected children failing first-line ART in a decentralised rural HIV programme. Methods Plasma samples were collected from 101 paediatric patients (⩽15 yrs of age) identified as failing ART. RNA was extracted from the plasma, reverse transcribed and a 1.3 kb region of the protease gene was amplified and sequenced using Sanger sequencing protocols. Sequences were edited in Geneius and drug resistance mutations were identified using the RegaDB and the Stanford, Rega and ANRS resistance algorithms. The prevalence and frequency of mutations were analysed together with selected clinical and demographic data in STATA v11. Results A total of 101 children were enrolled and 89 (88%) were successfully genotyped; 73 on a non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based regimen and 16 on a protease inhibitor (PI)-based regimen at the time of genotyping. The majority of patients on an NNRTI regimen (80%) had both nucleoside reverse-transcriptase inhibitor (NRTI) and NNRTI resistance mutations. M184V and K103N were the most common mutations amongst children on NNRTI-based and K103N among children on PI-based regimens. 23% had one or more thymidine analogue mutation (TAM) and 6% had ⩾3 TAMs. Only one child on a PI-based regimen harboured a major PI resistance mutation. Conclusions Whilst the patterns of resistance were largely predictable, the few complex resistance patterns seen with NNRTI-based regimens and the absence of major PI mutations in children failing PI-based regimens suggest the need for wider access to genotypic resistance testing in this setting. [ABSTRACT FROM AUTHOR]