This study characterized the antimicrobial resistance (AMR) genes present in a collection of 'Escherichia coli, Klebsiella' spp., and ' Staphylococcus aureus' isolates from bovine mastitis cases in Canada. Ampicillin-resistant 'E. coli' isolates along with second-generation cephalosporin resistant 'Klebsiella' spp., any MRSA, and a collection of penicillin-resistant 'S. aureus' isolates were screened for the presence of AMR genes and, in the case of 'S. aureus ', also virulence genes. A high diversity of [beta]-lactamase genes was detected in 'E. coli' and 'Klebsiella' spp. mastitis isolates in comparison to those found in beef cattle fecal isolates by other researchers, and the ubiquitous 'bla'CMY-2 gene was found in bacteria from mastitis. The [beta]-lactam-resistant ' S. aureus' isolates contained many 'blaZ' gene variants. The first MRSA isolate from bovine mastitis in Canada was identified as ST8, a sequence type normally associated with humans.