Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen of public health importance. In Chile, the Cordobes/Chilean clone was the predominant healthcare-associated MRSA (HA-MRSA) clone in 1998. Since then, the molecular epidemiological surveillance of MRSA has not been performed in Southern Chile. We aimed to investigate the molecular epidemiology of HA-MRSA infections in Southern Chile to identify the MRSA clones involved, and their evolutionary relationships with epidemic international MRSA lineages. A total of 303 single inpatient isolates of S. aureus were collected in the Valdivia County Hospital (2007-2008), revealing 33% (100 MRSA/303) prevalence for HA-MRSA infections. The SCCmec types I and IV were identified in 97% and 3% of HA-MRSA, respectively. All isolates lacked the pvl genes. A random sample (n = 29) of all MRSA was studied by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), SCCmec subtyping, agr and spa typing, and virulence genes profiling. PFGE analysis revealed the predominance (89%, 26/29) of pulsotype A and three additional pulsotypes, designated H1, I33, and G1. Pulsotype A (ST5-SCCmecI-spa-t149) is clonally related to the Cordobes/Chilean clone. Pulsotype H1 (ST5-SCCmecIVNT-spa-t002) is genetically related to the Pediatric clone (ST5-SCCmecIV). Pulsotype I33 (ST5-SCCmecIVc-spa-t002) is clonally related by PFGE to the community-associated MRSA (CA-MRSA) clone spread in Argentina, I-ST5-IVa-PVL(+). The G1 pulsotype (ST8-SCCmecIVc-spa-t024) is clonally related to the epidemic USA300 CA-MRSA. Here, we demonstrate the stability of the Cordobes/Chilean clone over time as the major HA-MRSA clone in Southern Chile. The identification of two CA-MRSA clones might suggest that these clones have entered into the healthcare setting from the community. These results emphasize the importance of the local surveillance of MRSA infections in the community and hospital settings.