The colonization of Staphylococcus aureus is one of the most important aggravating factors of atopic dermatitis (AD). Until now, the importance of S. aureus in AD and a positive correlation between colonization with S. aureus and clinical severity/skin barrier function has been demonstrated. The aim of this study was to determine whether there are certain clones of S. aureus which colonize the skin of AD patients. For this purpose, the genotype of S. aureus isolated from AD patients was examined by newly-developed typing methods. With 36 strains of S. aureus isolated from 35 patients with AD, spa typing, multi-locus sequence typing (MLST), and staphylococcal toxin gene assay by multiplex polymerase chain reaction, were performed. Clinical severity and skin barrier function were evaluated with eczema area and severity index (EASI) and with transepidermal water loss (TEWL). Among 36 strains of S. aureus, 14 sequence types (ST) and 20 spa types were identified, suggesting a very heterogeneous genetic composition of S. aureus and the absence of a prevailing genotype in S. aureus colonized with AD patients. Furthermore, there was no specific genotype of S. aureus which was associated with the clinical severity of AD or skin barrier dysfunction. A toxin gene assay, however, showed the predominance of S. aureus strains carrying sea and/or tsst-1. To the best of our knowledge, this is the first report to show the genetic composition of S. aureus strains isolated from AD patients determined by sequence-based typing methods.