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Background and Objective: Antibiotic-resistant Staphylococcus aureus strains have become a problem in treatment of infections caused by S. aureus. This study aimed to evaluate antibiotic resistance in S. aureus isolates from raw milk and detect femA gene in these isolates, as a confirmatory test for identification of S. aureus species. Methods: This cross-sectional study was performed on 110 raw milk samples. After culture in Cooked Meat broth, presence of S. aureus in grown colonies was confirmed in accordance with Iranian National Standard, No. 1194. Antibiotic resistance was then evaluated according to guidelines recommenced by the Clinical Laboratory Standards Institute. FemA-specific polymerase chain reaction was performed on antibiotic-resistant strains using specific primers and standard strains to differentiate S. aureus from other species. Results: S. aureus were found in 43 (39.09%) of the 110 collected samples. Among these isolates, 79.07% and 76.75% were phenotypically resistant to penicillin and ceftazidime, respectively. In addition, the femA gene was detected in all isolates. Conclusion: The results of this study show a high prevalence of resistance to penicillin and ceftazidime among S. aureus strains isolated from raw milk. [ABSTRACT FROM AUTHOR]