The present study was carried out to characterize Staphylococcus aureus isolated from subclinical cases of mastitis in cattle by PCR-RFLP using coagulase (coa) gene. In our study, 38 isolates of S. aureus recovered from Holstein-Friesian (H-F) crossbred and Rathi (a native breed) cattle with subclinical mastitis were subjected to coa gene amplification. Out of 38 isolates, 35 strains produced single amplicon of either 400, 490, 510, 550, 600, 710, 760, 810 or 850 bp while three isolates did not produce any coa amplicon. Subsequently, the products were digested with restriction enzyme AluI for typing of coa gene. Results showed higher coa gene polymorphism of S. aureus isolated from native breed (eight coa types) as compared to that in H-F crossbred cattle (three coa types). The Alu restriction endonuclease generated four and seven RFLP patterns with isolates from H-F crossbred and Rathi cattle respectively. The RFLP patterns obtained from similar amplicons in isolates from these two breeds did not show any variation.