Aim To evaluate the virulence determinants and genetic diversity of Staphylococcus aureus from bovine subclinical mastitis milk. Methods and Results PCR detection of virulence genes was performed for 173 Staph.aureus from bovine subclinical mastitis milk. Further, genetic diversity was analysed by agr and spa typing followed by pulsed field gel electrophoresis (PFGE) of selected isolates. Screening of virulence genes (n=19) showed the adherence genes viz. fnbA, clfA, fnbB and cna in 98 center dot 8, 97 center dot 1, 68 center dot 8 and 28 center dot 3 percentage of isolates, respectively, and 80 strains (46 center dot 24%) positive for enterotoxin genes were distributed as 23 toxinotypes, of which, 5 genotypes contained a single gene and the rest comprised of multiple toxin genes. Out of agr type-1 (87 center dot 3%), 74 center dot 2 per cent belonged to the three predominant spa types. Of 27 spa types, 11 were identified for the first time. The predominant spa types were t267 (N=44), t359 (N=42) and t6877 (N=29), which together accounts to 66 center dot 5 per cent of isolates. PFGE analysis of isolates (N=45) covering all the spa types revealed mostly similar or closely related pulsotypes. Local emergence of spa type t6877 in herd-dependant manner was observed. spa sequencebased phylogenetic analysis suggested t267 as the ancestral clone of t359, t6877 and other spa types except two. Conclusion Heterogenous virulence profile of the isolates had no significant association with the genotype. High prevalence of agr group I reaffirms their association with persistent subclinical mastitis. The spa type t267 appears to be the ancestral clone endemic in the region causing subclinical mastitis. In addition, few new spa types have emerged in the geographic region. Significance and Impact of Study Gives an insight into the genetic and evolutionary behaviour of Staph.aureus associated with bovine subclinical mastitis in India. The study would pave the way for devising effective control strategy for bovine mastitis in Indian context.