Children attending child care centers (CCCs) are at increased risk for infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Nasal colonization often precedes infection, and MRSA colonization has been associated with increased infection risk. Community-associated MRSA (CA-MRSA) has caused increased MRSA infections in the general population, including children. Little is known about the frequency of MRSA nasal colonization in young children, particularly in those attending CCCs where disease transmission is common. We sampled the nares of 1,163 children in 200 classrooms from 24 CCCs in North Carolina and Virginia to assess S. aureus colonization. MRSA strains were molecularly analyzed for staphylococcal cassette chromosome mec (SCCmec) type, Panton-Valentine leukocidin status, and multilocus sequence type. A case-control study was performed to identify risk factors for MRSA colonization. We found that 18.1% children were colonized with S. aureus and 1.3% with MRSA. Molecular analysis of the MRSA strains identified 47% as CA-MRSA and 53% as health care-associated MRSA (HA-MRSA). Although two centers had multiple children colonized with MRSA, genotyping indicated that no transmission had occurred within classrooms. The case-control study did not detect statistically significant risk factors for MRSA colonization. However, MRSA-colonized children were more likely to be nonwhite and to have increased exposure to antibiotics and skin infections in the home. Both CA-MRSA and HA-MRSA strains were found colonizing the nares of children attending CCCs. The low frequency of colonization observed highlights the need for a large multicenter study to determine risk factors for MRSA colonization and subsequent infection in this highly susceptible population.