Mastitis is a major dairy herd problem mainly caused by Staphylococcus aureus. The Staphylococcus aureus isolated strains from mastitic milk samples were subjected to molecular characterization of coa gene and phylogenetic tree (using MEGA6.1 software package) was constructed which revealed that the coa gene of S. aureus strains, could be grouped in two clades which were closely related to S. aureus isolates from Japan, India and Taiwan, while S. aureus isolates from Germany, UK and USA were distantly related. These results indicated the genetic relatedness of Pakistani isolates with other reported isolates from different parts of the world. All the Pakistani strains of coagulase gene were 99.98-100% similar while their divergence was 0.02%. The Pakistani strains were 99.9% similar and 0.07% divergent from Indian, Japanese, Taiwan, UK and German reported strains.These findings will also be helpful in future for designing suitable mastitis control strategies in the country.