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Staphylococcus aureus is a frequent and major contagious mastitis bacterial pathogen. The antibiotic treatment cure rates vary considerably from 4% to 92%. Staphylococcus aureus readily becomes resistant to antibiotics, resulting in persistent noncurable intramammary infection that usually results in culling of infected animals. Because of its notorious ability to acquire resistance to the commonly used as well as last resort antimicrobials such as methicillin and vancomycin and the development of multidrug-resistant strains, antimicrobial resistance (AMR) in S. aureus is of paramount importance in human medicine. The objective of this study was to evaluate the prevalence of AMR and genetic diversity of S. aureus isolates from milk of dairy cattle. Staphylococcus aureus isolates (n = 239) from 33 dairy farms in Tennessee were tested against 10 antimicrobials by broth microdilution method using the Sensititer system. Genetic diversity of resistant isolates was evaluated by pulsed-field gel electrophoresis (PFGE). Overall, AMR of the S. aureus isolates varied from as low as 1.3% for ceftiofur to as high as 25% for sulfadimethoxine. Out of 239 S. aureus isolates, 82 (34.3%) of them were resistant to at least 1 of the 10 antimicrobials. The AMR isolates belonged to two major PFGE types, indicating the presence of dominant clonal patterns among the resistant isolates. In general, there was a variation of prevalence of AMR within and among farms over time, with an increasing trend in tetracycline resistance. Judicious use of antimicrobials in dairy cattle farms can reduce the development of antimicrobial-resistant S. aureus.