Nosocomial infections involving epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains are a serious problem in many countries. In order to analyze outbreaks, the infectious isolates have to be typed; however, most molecular methods are expensive or labor-intensive. Here, we evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of cell extracts for the molecular characterization of S. aureus strains. The peak patterns of 401 MRSA and methicillin-susceptible S. aureus (MSSA) strains, including clinical and laboratory strains, were analyzed. Database searches indicated the peptides that were represented by the corresponding peaks in the spectra. The identities of the peptides were confirmed by the sequencing of mutants, the expression of antisense RNA fragments that resulted in the knockdown of the peptide of interest and the concomitant loss of the signal, or tandem MALDI-TOF MS (MALDI-TOF/TOF MS). It was shown that the signals derive mainly from stress proteins and ribosomal proteins. Peak shifts that differentiate the main S. aureus clonal complexes CC5, CC22, CC8, CC45, CC30, and CC1 correlate to point mutations in the respective genes. Retrospective typing of an MRSA outbreak showed that it is possible to differentiate unrelated MSSA, MRSA, and borderline resistant S. aureus (BORSA) strains isolated from health care workers. In conclusion, this method allows for the detection of the epidemic lineages of S. aureus during species identification by MALDI-TOF MS analysis.