Non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens have recently been used in HIV-1 infected children in resource-limited settings. Treatment failure with this regimen has become more common. A second regimen needs to be prepared for the Thai national program. Genotypic resistance testing was conducted among HIV-1 infected children who experienced virological failure with antiretroviral therapy (ART) using NNRTI-based regimens. Patterns of resistance mutations were studied and options for a second regimen were determined. There were 21 patients with a median (IQR) age of 4.1 (1.9-7.7) years. Sixteen patients were males. The median CD4 cell count and HIV-1 RNA at the time of virological failure were 647 cells/mm3 and 5.3 log copies/ml, respectively. The prevalences of patients with > or =1 major mutation conferring resistance to NRTIs and NNRTIs were 52% and 43%, respectively. Thymidine analoque mutations, M184V/I, and Q151M were observed in 38%, 33%, and 5%. The patterns of resistance mutations suggest that 48% of patients need a protease inhibitor-based regimen for the second regimen and didanosine+lamivudine is the most required nucleoside reverse transcriptase inhibitor backbone.