To assess the clonal structure of Staphylococcus aureus in the United States, we performed a molecular epidemiological study of 1,055 S. aureus isolates from a nationally representative clinical isolate collection from 2004–2008. Resistant and susceptible isolates were typed with multilocus sequence typing, tested for the presence of Panton-Valentine leukocidin (PVL), and serotyped. USA300 (multilocus sequence typing clonal complex 8, PVL positive, and methicillin-resistant) was the most frequently isolated clone, expanding from 12% of all isolates in 2004 to 38% in 2006. The USA300 clone increased significantly in frequency among both outpatients and inpatients. USA300 increased in both skin and soft-tissue and invasive infection isolates. The second most frequently observed clone was clonal complex 5, PVL-negative, and methicillin-resistant, and its frequency was stable from 2004–2008. The methicillin-susceptible S. aureus in the study was polyclonal, and decreased in frequency as it was replaced by USA300.