Development of an appropriate Staphylococcus aureus vaccine for bovine mastitis has eluded researchers for decades. The ability of S. aureus to form a protective exopolysaccharide capsule has posed a major obstacle because of the multiple serotypes and the poor immune response elicited by exopolysaccharides. This study characterized S. aureus serotypes isolated from cases of bovine mastitis obtained from veterinary diagnostic laboratories that service 44% of the dairy cattle in the United States. Major milk producing areas of the northeast, north central, Pacific coast and southwest were proportionately represented. Sub-samples of mastitic milk that contained S. aureus were frozen and sent to our laboratory for strain serotyping. The only other regional serotyping of S. aureus from bovine mastitis to date was done in France. The primary serotypes found were types 5 (51%) and 8 (18%) and 31% were non-typeable. In the current study, serotype 5 accounted for 18% of the isolates and serotype 8 for 23%. More importantly 59% of the isolates were not typeable with either type 5 or 8 antisera. These data indicate that S. aureus vaccines employing serotypes 5 and 8 would only be marginally effective in the United States. These data also suggest that development of a S. aureus vaccine for bovine mastitis should take into account regional variation in S. aureus serotypes.