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Objectives: To study the molecular characteristics, antibiogram and prevalence of multi-drug resistant Staphylococcus aureus (S. aureus) (MDRSA) isolated from milk obtained from culled dairy cows and from cows with acute clinical mastitis. Methods: Bacteria were cultured from 188 quarter milk samples obtained from cows before culling (n=139) and from cows affected with acute mastitis (n=49) belonging to 10 dairy farms. The bacteria were identified using colony morphology, Gram staining and biochemical characteristics. S. aureus isolates were then subjected to molecular charac-terization using PCR targeting 16S rRNA and mecA gene to identify Methicillin resistant S. aureus (MRSA). The antibiogram of all isolates was performed using the Kirby–Bauer disk diffusion method against 10 commonly used antibiotics in dairy farms. Results: S. aureus was isolated from 19 (13.7％) samples obtained from culled cows and 11 (22.4％) samples obtained from cows with acute mastitis. In both culled cows and cows with acute mastitis, in vitro antibiogram revealed that 100％ of S. aureus isolates were resistant to erythromycin, penicillin G, streptomycin, doxycyclin, and trimethoprim/sulpha. The prevalence of MRSA in milk of culled cows and cows with acute mastitis was 26.3％ and 18.2％, respectively, with an overall prevalence of 3.7％ among all samples. All MRSA isolates were completely resistant to all tested antibiotics. All MRSA isolates were positive for the presence of the mecA gene. Conclusions: MRSA carrying the mecA gene were isolated from mastitic milk from dairy cows in Jordan for the first time. MRSA may pose a potential health risk to the public, farm workers and veterinarians.