OBJECTIVE The use of a potent combination of antiretroviral (ARV) drugs, so-called highly active ARV therapy (HAART), has dramatically improved the quality of life and overall survival of children with human immunodeficiency virus (HIV) infection. However, these benefits can be compromised by the development of drug resistance. Our objectives were to analyze the prevalence and pattern of HIV-drug resistance among HIV-infected children failing first-line HAART. METHODS Retrospective study based on data obtained from July 2006 through January 2009 of prevalence of genotypic resistance estimated in HAART-treated children who experienced virologic failure (HIV RNA > 1000 copies/mL) at a tertiary care center in Riyadh. METHODS The characteristics of the study population and genotype resistance data were analyzed in ARV-treated children who experience virologic failure. RESULTS Among 22 children who underwent resistance testing, the prevalence of resistance to any drug was 86.4%. Inadequate adherence to ARVs in children with drug resistance was 91%. Twenty-four mutations were detected within the protease coding region and 14 in the reverse transcriptase (RT) coding region. In 80% of isolates piM36I was detected, while rtM184V was detected in 70% of the isolates and was associated with cross-resistance to at least two nucleoside RT inhibitors (NRTI). Clinically significant non-nucleoside RT inhibitors (NNRTI) resistance was conferred by rtK103N. The best ARV susceptibility was to lopinavir in the PI class. ARV resistance was not associated with geographic regions or the CDC classification status. Study children responded satisfactorily to genotype-guided treatment and intensive family counseling. CONCLUSIONS ARVs resistance is common among HIV-infected Saudi children who experienced virologic failure to HAART. Inadequate adherence is a common cause for resistance to ARVs in children. Mutations M36I and M184V were more frequent for PIs, NRTIs and NNRTIs. Evaluation of genotype tests should be considered in all children with therapeutic failure to guide future selection of ARV regimens. These data will help improve clinical management of HIV-infected children in Saudi Arabia.