•No MRSA was detected among bovine mastitis-associated Staphylococcus aureus isolates in Australia.•Over all low levels of antimicrobial resistance was detected. Resistance to only penicillin (12.4%) and erythromycin (0.5%) was identified among bovine mastitis-associated S. aureus isolates in Australia.•Most bovine mastitis-associated S. aureus belong to clonal complexes 97, 705, and 1.•Comparative genomic analysis revealed that Australian bovine S. aureus isolates clustering together according to their ST separately from human isolates.•S. aureus ST352 phylogeny provided evidence for geographical clustering of Australian strains into a distinct grouping separate from international bovine isolates.•Australian S. aureus isolates have limited genetic diversity and are genetically distinct from human and international bovine S. aureus isolates. The aim of this study was to investigate antimicrobial resistance and population structure of bovine mastitis-associated Staphylococcus aureus isolates, and compare them to human isolates obtained from Western Australian hospitals and overseas strains to determine relatedness to human isolates from a zoonotic or reverse zoonotic aspect. Antimicrobial susceptibility testing was performed on 202 S. aureus isolates of which 166 isolates underwent whole genome sequencing. Only resistance to penicillin (12.4%) and erythromycin (0.5%) was identified and of note, no resistance was demonstrated to oxacillin. Genomic characterisation identified 14 multilocus sequence types (STs), with most isolates belonging to clonal complexes 97, 705, and 1. Four distinct clades based on virulence gene composition were identified. The four clades were predominantly ST based, consisting of ST352, ST97, ST81/ST1, and ST705. Core genome comparison of the bovine and human S. aureus isolates demonstrated defined clustering by ST, with the Australian bovine S. aureus isolates clustering together according to their ST separately from human isolates. In addition, a bovine specific cluster comprising Australian ST151 and ST705 isolates, and ST151 isolates from Irish dairy cattle was clearly delineated. Examination of a detailed ST352 phylogeny provided evidence for geographical clustering of Australian strains into a distinct grouping separate from international strains. This study has identified Australian S. aureus isolates have limited genetic diversity and are genetically distinct from human and international bovine S. aureus isolates. Current first line therapies for bovine mastitis in Australian dairy cattle remain appropriate.