To determine the strain variation and persistence among isolates of methicillin-resistant Staphylococcus aureus (MRSA) cultured from patients with colonization over extended time spans, pulsed-field gel electrophoresis was used to analyze the isolates from 47 patients for whom at least two mecA-positive isolates collected a minimum of six months apart were available. For 22 (47%) patients, the isolates represented multiple distinct strains of Staphylococcus aureus, while 20 (43%) patients had only a single strain detected; five (11%) patients had similar, genetically related isolates. MRSA were frequently associated with mucocutaneous abnormalities; 29 (62%) patients had focal cutaneous defects, and ten (21%) had chronic dermatitis. Multiple strains of MRSA were detected more frequently than single strains among patients in whom the initial focus of MRSA resolved clinically and another mucocutaneous defect subsequently developed compared to patients with clinically persistent foci (11/15 versus 9/23, respectively; p = 0.05, Fisher's exact test). Among the 21 patients in this series for whom isolates cultured within a two-month time span were available, there were seven (33%) patients with multiple strains of MRSA, including one patient with polyclonal bacteremia. In summary, patients with long-term MRSA colonization often have several different strains of MRSA, which typically change overtime in association with removal or resolution of a colonized focus and the recurrence of mucocutaneous defects.