Background: Limited data are available on patterns of resistance mutations in pediatric patients in southern Africa, where HIV-1 subtype C (HIV-1C) predominates. Methods: Retrospective chart review of pediatric patients. Nucleoside reverse transcriptase inhibitor (NRTI)-and nonnucleoside reverse transcriptase inhibitor (NNRTI)-associated resistance mutations quantified from population-based sequencing genotypic resistance assay results taken at time of first-line antiretroviral therapy (ART) failureTotal number of patients with resistance assays analyzed is 45. Nucleoside reverse transcriptase inhibitor-associated mutation frequencies noted were M184V (n ¼ 41; 91.1%); thymidine analogue mutations (TAMs; n ¼ 20; 44.4%); >1 TAM (n ¼ 9; 20%); TAM-2 pathway (n ¼ 10; 22.2%); TAM-1 pathway (n ¼ 7; 15.6%); TAM-1 and TAM-2 pathways (n ¼ 3; 6.7%); K65R (n ¼ 2; 4.4%); Q151M (n ¼ 1; 2.2%); and L74V (n ¼ 0; 0%). Nonnucleoside reverse transcriptase inhibitor-associated mutation frequencies noted were associated with notable resistance to either/both NVP and EFV (n ¼ 40; 88.9%); K103N (n ¼ 15; 33.3%); 1 mutations associated with etravirine (ETR) failure (K101E, Y181C, and G190A; n ¼20; 44.4%); and 2 notable NNRTI mutations (n ¼ 12; 26.7%). Conclusions: In this cohort, low-genetic barrier mutations were common, as were TAMs, including more than 1 TAM. Mutations compromising nonthymidine analogue backbones were rare, suggesting that it is likely that children who fail firstline NRTI backbones containing d4T or ZDV/3TC would still respond to abacavir (ABC), didanosine (ddI), and, for adolescents, tenofovir (TDF). Our data support the empiric continuation of 3TC in second-line regimens.