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Staphylococcus aureus is a known pathogen, causing serious food-borne intoxications due to the production of enterotoxins, being otherwise a major cause of mastitis. In this sense, the detection of S. aureus is an important issue for the food industry to avoid health hazards and economic losses. The present work applied MALDI-TOF MS for the classification of 40 S. aureus strains, 36 isolated from Italian dairy products and four from human samples. All isolated strains were clearly identified as S. aureus by their spectral fingerprints. The peak masses m/z 3444, 5031, and 6887 were determined to be specific biomarkers for S. aureus. Furthermore, clustering of the peak mass lists was successfully applied as a typing method, resulting in eight groups of strains. This is the first time that a detailed spectral comparison was carried out and characteristic peak masses were determined for every spectral group. Three strains exhibited a peak at m/z 6917 instead of m/z 6887, which was related to four polymorphisms in their 16S rRNA sequences. However, the grouping obtained by MALDI-TOF MS fingerprinting could not be related to toxin production or to the origin of the strains., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)