Mastitis is an important disease in dairy herds, typically caused by bacterial infection such as Staphylococcus aureus. Antibiotic resistance in S. aureus is worldwide public health problem and considerable concern that continues to grow. The objective of the present study was to investigate the antibiotic susceptibility of S. aureus strains by disk diffusion test and determination of mecA, ermA, ermB, ermC, and msrA antibiotic resistance genes of this bacterium isolated from cow’s milk using multiplex PCR method. A total of 100 cow’s milk samples that suspected for mastitis were collected from traditional centers and cattle industries in the Chaharmahal Va Bakhtiari province (Southwest Iran). All specimens were cultivated in sheep blood agar (SBA) for isolation of S. aureus and then catalase, oxidase, and the coagulase tests were carried out. Bacterial DNA was extracted from colonies cultivated onto sheep blood agar using DNA extraction kit. Then multiplex PCR performed by specific primers for detection of antibiotic resistance genes of S. aureus and amplified products were separated on 1% agarose gel electrophoresis. The electrophoresis revealed 163, 174, 139, 224, and 190 bp(s) for msrA, mecA, ermA, ermB, and ermC, genes, respectively. Multiplex PCR assay showed that 35 (41.18%), 25 (29.41%), 20 (23.53%), 17 (20%), and 16 (18.82%) of S. aureus samples, contained msrA, mecA, ermA, ermB, and ermC, genes, respectively. Furthermore, msrA gene is more frequent than other antibiotic resistance genes. These results indicated that msrA gene could be related to increasing of MLS antibiotics resistant in S. aureus and the findings of this study could be useful for foodstuff, public health, and dairy industry to decrease S. aureus antibiotic resistance strains. So, these data may be helpful for prescription of best drugs for control of the infection caused by this bacterium in cows for the reduction of mastitis.