This study aimed to evaluate the incidence of Staphylococcus aureus infections in different departments of Belcolle Hospital in Viterbo and the surrounding area between January 2003 and June 2008. Isolates of methicillin-resistant S. aureus (MRSA) recovered in this time interval were characterized by microbiological and molecular methods to evaluate the reliability of simple criteria to distinguish between hospital-acquired and community-acquired isolates. MRSA accounted for 33% of all S. aureus, with a significantly higher prevalence in isolates from nosocomial infections. MRSA isolates were assayed by PCR for the presence of 13 genes associated with virulence, agr type and SCCmec type. Cumulative data were analysed by partial least square discriminant analysis and a clear correlation was demonstrated between genetic profiles and classification of isolates as hospital or community acquired according to simple temporal criteria. Nosocomial MRSA isolates from blood samples showed significantly higher genetic diversity than other nosocomial isolates. Our data confirm the existence of significant differences between community- and hospital-acquired MRSA isolates.