Intrapartum administration of single-dose nevirapine (sdNVP) reduces perinatal HIV-1 transmission in resource-limiting settings by half. Yet this strategy has limited effect on subsequent breast milk transmission, making the case for new treatment approaches to extend maternal/infant antiretroviral prophylaxis through the period of lactation. Maternal and transmitted infant HIV-1 variants frequently develop NVP resistance mutations following sdNVP, complicating subsequent treatment/prophylaxis regimens. However, it is not clear whether NVP-resistant viruses are transmitted via breastfeeding or arise de novo in the infant.We performed a detailed HIV genetic analysis using single genome sequencing to identify the origin of drug-resistant variants in an sdNVP-treated postnatally-transmitting mother-infant pair. Phylogenetic analysis of HIV sequences from the child revealed low-diversity variants indicating infection by a subtype C single transmitted/founder virus that shared full-length sequence identity with a clonally-amplified maternal breast milk virus variant harboring the K103N NVP resistance mutation.In this mother/child pair, clonal amplification of maternal NVP-resistant HIV variants present in systemic and mammary gland compartments following intrapartum sdNVP represents one source of transmitted NVP-resistant variants that is responsible for the acquisition of drug resistant virus by the breastfeeding infant. This finding emphasizes the need for combination antiretroviral prophylaxis to prevent mother-to-child HIV transmission.