ABSTRACT The present study was carried out for pheno-genotypic characterization of L. monocytogenes (L. monocytogenes). A total of three isolates of L. monocytogenes were recovered from 85 mastitic milk samples (47 buffalos and 38 cows). Confirmation of the L. monocytogenes were based on biochemical tests followed by phenotypic characterization by hemolysis on sheep blood agar, the Christie Atkins Munch-Petersen (CAMP) test, phosphatidylinositol-specific phospholipase C (PIPLC) assay and phosphatidylcholine-specific phospholipase C (PIPLC) assay. The isolates were subjected to genotypic characterization with the help of PCR assay for five virulence associated genes namely, plcA, prfA, hlyA, actA and iap. The L. monocytogenes isolates were further subjected to multiplex-PCR based serotyping. All the three isolates of L. monocytogenes were hemolytic, CAMP positive, PI-PLC, PC-PLC positive, hlyA, pclA, actA, iap and prfA positive by PCR. All the three isolates of L. monocytogenes were serotyped as 4b