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To quantify primary resistance mutations (PRMs) among HIV-1-infected women receiving antiretroviral therapy (ART) for prevention of mother-to-child transmission (MTCT).Peripheral blood mononuclear cell samples from HIV-1-infected women enrolled in a prospective cohort study in Argentina, the Bahamas, Brazil, and Mexico (NISDI Perinatal Study) were assayed for PRMs. Eligible women were those enrolled by March 2005 and diagnosed with HIV-1 infection during the current pregnancy, and who received ART for MTCT prophylaxis and were followed for 6–12 weeks postpartum.Of 819 women, 198 met the eligibility criteria. At enrollment, 98% were asymptomatic, 62% had plasma viral load < 1000 copies/ml, 53% had CD4+ cell count ≥ 500 cells/μl, and 78% were ART-exposed (mean duration, 8.0 weeks; 95% confidence interval, 7.1–8.9). The most complex ART regimen during pregnancy was usually (81%) a three-drug regimen [two nucleoside reverse transcriptase inhibitors (NRTIs) + one protease inhibitor or two NRTIs + one non-nucleoside reverse transcriptase inhibitor). PRMs were observed in samples from 19 (16%) of 118 women that were amplifiable at one or both time points [11/76 (14%) at enrollment; 14/97 (14%) at 6–12 weeks]. The occurrence of PRMs was not associated with clinical, immunological, or virological disease stage at either time point, whether ART-naive versus exposed at enrollment, or the most complex or number of antiretroviral drug regimens received during pregnancy (P > 0.1). Of 55 women with amplifiable samples at both time points, PRMs were detected in 11 samples (20%).PRMs occurred among 16.1% of relatively healthy HIV-1-infected mothers from Latin American and Caribbean countries receiving MTCT prophylaxis.