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We carried out a nationwide study aimed at the determination of the molecular epidemiology and antibiotic resistance mechanisms of invasive Staphylococcus aureus in 21 Spanish hospitals.The distributions of molecular markers, including antibiotic resistance genes, were investigated in 203 S. aureus, comprising 90 methicillin-resistant S. aureus (MRSA) and 113 methicillin-susceptible S. aureus (MSSA). Antimicrobial susceptibility was determined by standard methods. Panton–Valentine leucocidin (PVL) detection, staphylococcal cassette chromosome mec (SCCmec) types and agr types were performed/determined by PCR. All isolates were genotyped by PFGE after digestion of chromosomal DNA with SmaI. Multilocus sequence typing and spa-typing were also performed.In MRSA isolates, 74.4% were agr allotype II and were positive for SCCmec IV. Sixty-nine spa-types were identified, 18 in MRSA and 57 in MSSA. Both MRSA and MSSA variants were detected in six spa-types (8.7%). The majority of S. aureus (51.2%) were grouped into four spa-types (t067, t002, t012 and t008). The spa-type t067 was detected in 18 of the 21 (85.7%) participating hospitals, including both MRSA and MSSA in six of them; in total, 25.9% of our isolates were spa-type t067 (49% in MRSA) in comparison with 0.6% in a central spa-typing database. The prevalence of the ant(4′)-Ia and msrA/msrB genes was significantly higher in the MRSA spa-type t067 than in the other MRSA spa-types. Association between spa-type t067 and ST125 is described here for the first time. A high prevalence (36.4%) of PVL-positive MSSA was detected.A higher than expected prevalence of spa-type t067 isolates was found among invasive MRSA in Spain. The oxacillin, tobramycin, erythromycin and ciprofloxacin resistance profile of spa-type t067 isolates was linked to the presence of ant(4′)-Ia and msrA or msrB genes.