The co-existence of multiple genotypes in colonization by Staphylococcus aureus has not been fully investigated. The aim of this study was to evaluate the heterogeneity of S. aureus carriage in children. We evaluated 125 nasal and perianal swab samples that were positive for S. aureus from 76 children scheduled for elective surgery. For each sample, at least four colonies with the same or different morphotypes were selected for analysis. Multiple-locus variable-number tandem-repeat fingerprinting was used to determine the genetic relatedness and to characterize the clonality of the S. aureus strains. Of the 125 swabs, 91 (73 %) contained meticillin-sensitive S. aureus (MSSA), 8 (6 %) contained meticillin-resistant S. aureus (MRSA), and 26 (21 %) contained MSSA and MRSA simultaneously. A total of 738 S. aureus strains were evaluated with a mean of 6 colonies (range 4-15) picked from each culture. Of the 125 swabs, 32 (26 %) samples contained two genetically distinct S. aureus strains and 6 (5 %) contained three different genotypes. Multiple S. aureus strains simultaneously carried by individual children were genetically unrelated to each other. We concluded that the co-existence of multiple genotypes of S. aureus was common. The significance of multiple carriage is yet to be determined, but this intraspecies interplay could be important to pathogenicity and virulence in S. aureus.