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Background: Single-dose nevirapine (sd-NVP) has been the main option for prevention of mother-to-child transmission (PMTCT) of HIV-1 in low-resource settings. However, sd-NVP can induce the selection of HIV-1 resistant mutations in mothers and infants. In Mozambique, there are limited data regarding the profile of NVP resistance associated mutations (RAM) in the context of PMTCT. Objectives: To assess the prevalence and the factors associated with NVP RAM among children born to HIV-1 infected mothers enrolled in the PMTCT programme adopted in Mozambique. Methods: One hundred and fifty seven children aged 6 to 48 weeks were sequentially included (July 2011 to March 2012) at four centres in Maputo. Genotyping of RAM was performed in samples with HIV-1 RNA≥ 100 copies/μL (Viroseq). Sequencing was performed with ABI 3100 (Applied Biosystems). Logistic regression modelling was undertaken to identify the factors associated with NVP RAM. Results: Seventy-nine children had their samples genotyped. Their median age was 7.0 (3–12) months and 92.4% received prophylaxis with sd-NVP at birth plus daily NVP. 35.4% of mothers received antiretrovirals (ARVs) for PMTCT. ARV RAM were detected in 43 (54.4%) of the children. 45.6% of these children had at least one NVP RAM. The most common mutations associated with NVP resistance were K103N (n = 16) and Y181C (n = 15). NVP RAM was significantly associated with mother exposure to PMTCT (crude odds ratio [OR] 30.3, 95% CI 4.93–186.34) and with mother’s CD4 count < 350 cells/mm3 (crude OR 3.08, 95% CI 1.02–9.32). In the multivariable analysis the mother’s exposure to PMTCT was the only variable significantly associated with NVP RAM (adjusted OR 48.65, 95% CI 9.33–253.66). Conclusions: We found a high prevalence of NVP RAM among children who were exposed to the drug regimen for PMTCT in Mozambique. The mothers’ exposure to PMTCT significantly increased the risk of NVP RAM. [ABSTRACT FROM AUTHOR]