We sought to determine the pattern of resistance-associated mutations (RAMs) among HIV-1-infected children failing first-line antiretroviral therapy (ART) and ascertain their response to second-line regimens in 48 weeks of follow-up. The design involved a cohort study within an HIV care program. We studied records of 142 children on ART with virological failure to first-line ART and switched to second-line ART with prior genotypic resistance testing. The pattern of RAMs was determined in frequency runs and the factors associated with accumulation of≥3 thymidine analogue mutations (TAMs) and K103N were determined using multivariate logistic models. Changes in weight, height, CD4, and viral load at weeks 24 and 48 after switch to second-line therapy were determined using descriptive statistics. The children were mean age 10.9±4.6 years and 55.6% were male. The commonest nucleoside reverse transcriptase inhibitor (NRTI) RAM was M184V in 129/142 (90.8%) children. TAMs,≥3 TAMs, 69 insertion complex, K65R/N, and Q151M were observed in 43.0%, 10.6%, 18.3%, 2.8%, and 2.1% of the children, respectively. The commonest nonnucleoside reverse transcriptase inhibitor (NNRTI) RAM was K103N in 72/142 (50.7%) children. The starting ART regimen was associated with accumulation of both≥3 TAMs (p=0.046) and K103N (p<0.0001), while a history of poor adherence was associated with K103N accumulation (p=0.0388). After 24 weeks and 48 weeks of follow-up on lopinavir-ritonavir based second-line ART, 86/108 (79.6%) and 84.5% (87/103) of the children had viral loads<400 copies/ml, respectively. The mean CD4 absolute count increased by 173 cells/μl and 267cells/μl at weeks 24 and 48, respectively. Increments were also observed in mean weight (1.6 kg and 4.3 kg) and height (1.8 cm and 5.8 cm) at weeks 24 and 48, respectively. Multiple RAMs were observed among HIV-1-infected children with virological failure on first-line ART with M184V and K103N most frequent. The children responded favorably to boosted PI-based second-line ART.