ABSTRACT In 2005, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) anovaginal colonization in pregnant women at our center (Columbia University Medical Center) was 0.5%, and MRSA-colonized women were less likely to carry group B streptococcus (GBS). In this study, our objectives were to identify changing trends in the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) anovaginal colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to characterize the MRSA strains. From February to July 2009, Lim broths from GBS surveillance samples were cultured for S. aureus . MRSA strains were identified by resistance to cefoxitin and characterized by MicroScan, staphylococcal cassette chromosome mec (SCC mec ) typing, pulsed-field gel electrophoresis (PFGE), spa typing, and Panton-Valentine leukocidin PCR. A total of 2,921 specimens from different patients were analyzed. The prevalences of MSSA, MRSA, and GBS colonization were 11.8%, 0.6% and 23.3%, respectively. GBS colonization was associated with S. aureus colonization (odds ratio [OR], 1.9; 95% confidence interval [95% CI], 1.5 to 2.4). The frequencies of GBS colonization were similar in MRSA-positive (34.2%) versus MRSA-negative patients (21.8%) ( P = 0.4). All MRSA isolates from 2009 and 13/14 isolates from 2005 were SCC mec type IV or V, consistent with community-associated MRSA; 12/18 (2009) and 0/14 (2005) isolates were the USA300 clone. Levofloxacin resistance increased from 14.3% (2005) to 55.6% (2009) ( P = 0.028). In conclusion, the prevalence of MRSA anovaginal colonization in pregnant women in New York City, NY, remained stable from 2005 to 2009, and USA300 emerged as the predominant clone with a significant increase in levofloxacin-resistant isolates.