Introduction: There are various virulence factors encoded by Staphylococcus Aureus, which enable them to cause nosocomial infections and mastitis in dairy cattle. The CLF-A gene mediates the bacterial colonization through binding to the extracellular matrix of the host. Treatment of such infections becomes more difficult due to increased resistance to methicillin. The current study aimed at investigating the frequency of CLF-A, mec-A, and mec-C genes inS. Aureus strains isolated from hospital infections and cow’s milk.Methods: A total of 280 clinical samples as well as 100 milk samples from cattle with mastitis were collected. After identification of isolates, methicillin-resistant strains were identifies using agar screening and the cefoxitin disc diffusion test. In addition, the Minimum Inhibitory Concentration (MIC) value of oxacillin was determined using the E-test method. Polymerase Chain Reaction (PCR) was used to detect CLF-A, mec-A, and mec-C genes in the methicillin-resistant isolates. Out of 120S. Aureus strains isolated from nosocomial infections, 40 were identified as Methicillin-Resistant Staphylococcus Aureus (MRSA), while only 1 isolate from cow’s milk was MRSA.Results: Out of the 280 studied isolates, 35 clinical strains (87.5%) carried mec-A gene, and the frequency of CLF-A gene was 80% and 100% in clinical and bovine milk samples, respectively.Importantly, MRSA strains harbouring CLF-A gene, isolated from wound samples, exhibited the highest frequency. The mec-C gene was not found in clinical and milk isolated strains. The high frequency of CLF-A gene in MRSA strains isolated from wound indicated a probable role of this virulence factor in skin colonization, as well as distribution and spread of the strains.Conclusion: Development of an appropriate method seems to be particularly useful for preventing the distribution of such strains.