Background. The HIV Network for Prevention Trials (HIVNET) 012 trial showed that NVP resistance (NVPR) emerged in some women and children after the administration of single-dose nevirapine (SD-NVP). We tested whether K103N-containing human immunodeficiency virus (HIV)-1 variants persisted in women and infants 1 year or more after the administration of SD-NVP. Methods. We analyzed samples from 9 women and 5 infants in HIVNET 012 who had NVPR 6-8 weeks after the administration of SD-NVP. Samples were analyzed with the ViroSeq system and with 2 sensitive resistance assays, LigAmp and TyHRT. Results. ViroSeq detected the K103N mutation in 8 of 9 women and in 2 of 5 infants. LigAmp detected the K103N mutation at low levels in 8 of 9 women and in 4 of 5 infants. K103N was not detected by ViroSeq 12-24 months after the administration of SD-NVP but was detected by LigAmp in 3 of 9 women and in 1 of 5 infants. K103N was also detected in those samples by use of the TyHRT assay. Conclusions. K103N-containing variants persist in some women and infants for 1 year or more after the administration of SD-NVP. Sensitive resistance assays may provide new insight into the impact of antiretroviral drug exposure on HIV-1 evolution.