ABSTRACT This study aimed to assess the antimicrobial susceptibility of 52 Staphylococcus aureus (S. aureus) bovine mastitis isolates obtained from 37 dairy herds from the northwest of Portugal against antibiotics belonging to the β-lactam family, evaluate the detection of blaZ and mecA resistance genes, and study the diversity of positive isolates. The antimicrobial susceptibility tests were performed by the disk diffusion method. The detection of blaZ and mecA genes was performed using specific polymerase chain reaction (PCR) and the diversity of blaZ was evaluated by phylogenetic analysis. The antimicrobial susceptibility test showed a prevalence of phenotypic resistance by S. aureus of 52.0% against penicillin and ampicillin, 34.6% against oxacillin, 17.3% against amoxicillin plus clavulanic acid, and 21.1% against cefazolin. A prevalence of phenotypic intermediate resistance of 5.7% against penicillin, 9.6% against amoxicillin plus clavulanic acid, and 1.9% against ampicillin and cefazolin, respectively, was demonstrated. A 100.0% phenotypic susceptibility was found against piperacillin. Of the 52 S. aureus isolates, 35 (67.3%) were PCR positive for blaZ gene, and isolate 25 was positive for mecA gene. The phylogenetic analysis of partial blaZ gene S. aureus isolates consensus sequences were placed in two different clades, clade A (cluster A, A.1) and B (cluster B), all closely related to animal and/or human S. aureus strains. Isolate 2 appeared in the phylogenetic tree as the most divergent. This study indicates that blaZ resistance gene plays a role in β-lactam resistance in the tested bovine mastitis S. aureus isolates within dairy herds in the northwest of Portugal, especially in case of penicillin and ampicillin antibiotics that have shown a high phenotypic prevalence. Indeed, the proportion of bovine mastitis isolates with phenotypic resistance did not agree with the proportion of those identified for blaZ, as isolates with 100.0% of phenotypic susceptibility for all tested antibiotics also harbored blaZ. BlaZ phylogenetic analysis from S. aureus isolates showed diversity inside or between different herds in the northwest of Portugal. Piperacillin, as a suggestion, could be tested for S. aureus bovine mastitis treatment in the future to evaluate this new possibility of therapy. 5 S (S 3 βlactam β lactam family method (PCR 520 0 52.0 346 34 6 34.6 oxacillin 173 17 17.3 acid 211 21 1 21.1 cefazolin 57 7 5.7 96 9 9.6 19 1.9 respectively demonstrated 1000 100 100.0 piperacillin 67.3% 673 67 (67.3% clades cluster A.1 A1 B, , B) andor strains divergent Indeed Piperacillin suggestion therapy 52. 34. 17. 21. 5. 9. 1. 10 100. 67.3 (67.3 A. 67. (67. (67 (6 (