This study evaluated the genetic diversity and antimicrobial susceptibility of Staphylococcus aureus isolates from dairy cows in Minas Gerais, Brazil. Thirty-seven isolates from five municipalities (8 herds) were genotyped using multi-locus sequence typing (MLST) and susceptibility to 12 antimicrobial agents was tested using the disk diffusion method. High resistance rates for penicillin [75.68% (28/37)], ampicillin [70.27% (26/37)], and tetracycline [70.27% (26/37)] were detected. Multidrug resistance was observed in seven [18.92% (7/37)] isolates, and two were suggestive of Methicillin-resistant Staphylococcus aureus (MRSA). Among the 37 isolates, 33 novel sequence types (ST) and two known STs (ST126 and ST746) were identified in MLST. The clonal complexes more frequently observed were: CC97 [78.38%; (29/37)], CC1 [8.11%; (3/37)] and CC5 [5.40%; (2/37)]. Minimum‐spanning tree (MST) analysis according to data from municipalities, herds, and resistance patterns for all isolates did not show any clustering pattern. However, the MST comparing all Brazilian S. aureus isolates deposited in the PubMLST database and from this study depicted an association between the genotype and strain origin (clinical sample). Isolates from this study that belong to CC97 were close to database isolates from milk and dairy products, while those that belong to CC1 and CC5 were close to database isolates from human sources and the environment of dairy farms or industries. In conclusion, our results showed a high rate of resistance to penicillins and tetracyclines and great genetic diversity among the S. aureus isolates from bovine mastitis genotyped in the present study.