OBJECTIVE: To estimate the frequency of genital tract colonization by methicillin-resistant Staphylococcus aureus (MRSA) among pregnant women and evaluate the association of such colonization with infant outcome. METHODS: Between July 2003 and July 2006, anovaginal screening cultures for group B Streptococcus (GBS) were prospectively obtained in the third trimester (35 to less than 37 weeks of gestation) and were also processed for identification of Staphylococcus aureus including methicillin-resistant strains. Maternal colonization by MRSA was linked to a computerized database of invasive neonatal infections that occurred at our center during the study period. RESULTS: Among 5,732 mothers (who delivered 5,804 infants) with GBS screening cultures and infant infection data available, 22.9% were GBS-positive and 14.5% were positive for Staphylococcus aureus. A total of 24.3% of the Staphylococcus aureus isolates were MRSA. The overall MRSA colonization rate was 3.5%. Colonization by any Staphylococcus aureus (relative risk 1.6, 95% confidence interval 1.4-1.9) as well as MRSA (relative risk 2.2, 95% confidence interval 1.6-2.8) was significantly more common among GBS-positive than among GBS-negative women. No cases of early-onset invasive neonatal infection by MRSA occurred among infants in the study. CONCLUSION: Genital tract colonization with MRSA affected 3.5% of pregnant women. Such MRSA colonization is associated with colonization by GBS but does not predispose to a high risk of early-onset neonatal MRSA infection. © 2008 The American College of Obstetricians and Gynecologists.