In this study, 225 milk samples were collected sequentially during 1 st to 88 th day from 25 HF cross cows in an organized farm. First five collections were obtained at a weekly interval (1,7,14,21 and 28 days) and later, fortnightly for two months (43, 58, 73 and 88 days). These milk samples were screened for Subclinical mastitis (SCM) by Somatic Cell Count (SCC). Further, multiplex-PCR for detection of S.aureus, E.coli, S.agalactiae, S.dysgalactiae and S.uberis was employed to detect the major bacterial pathogens. The SCM positivity was assessed based on criteria of SCC ≥ 500,000 cells /ml. The study revealed the high prevalence of variable SCM pattern in milking cows by SCC (73.33 %) in sequentially collected milk samples over a period of 88 days. No specific pattern of prevalence of SCM was observed during the study period. The prevalence of SCM was not influenced by the stage of lactation. In all the stages of lactation and age groups S. aureus, Streptococci and E.coli were detected with the predominance of S. aureus. The varied distribution of organisms in different stages of lactation did not influence the prevalence of SCM. Further, the high prevalence of SCM was noticed in aged cows. Among these, maximum number of milk samples (46 %, 52/113) revealing the presence of pathogens were obtained from cows in the age group 7-11 years. The multiplex PCR was found an easy and rapid method to detect the predominant pathogens causing SCM. The findings emphasize the need to control SCM through sequential monitoring of SCM through SCC, multiplex-PCR and proper managemental practices.