The present study was conducted on 102 Sahiwal dairy cows to assess the prevalence of subclinical mastitis, type of bacteria present and ability of different parameters to diagnose subclinical mastitis. The prevalence of subclinical mastitis was found to be 43.12% animal wise and 18.14% quarter wise. The coagulase negative staphylococci (CNS) were the most common isolate both in specific mastitis (59%) and latent infections (47%). The Corynebacterium was found another important isolate in latent cases (33%). The evaluation of different markers in the diagnosis of subclinical mastitis revealed N-acetyl-beta-D-glucosaminidase (NAGase) as the most sensitive (84.29%) and the California Mastitis Test (CMT) most specific (89.09%) test. The milk SCC showed a good correlation with CMT (0.68) and NAGase (0.59). The CMT and NAGase possessed a correlation coefficient of 0.60. Overall, the CMT, NAGase and electrical conductivity (EC) were found as best markers for identification of subclinical mastitis quarters in Sahiwal cows.