A total of 1002 samples comprising blood (n = 248), faecal swabs (n = 248), nasal swabs (n = 248) and deep vaginal swabs (n = 248) collected from the 248 sheep and 10 environmental samples of 10 sheep flocks were examined for the presence of pathogenic Listeria spp. Confirmation of the isolates was based on biochemical tests followed by phenotypic characterization by haemolysis on sheep blood agar, Christie Atkins Munch-Petersen (CAMP) test phosphatidylinositol-specific phospholipase C (PI-PLC) assay and phosphatidylcholine-specific phospholipase C (PI-PLC) assay. The isolates were subjected to genotypic characterization with the help of PCR assay for five virulence-associated genes, plcA, prfA, hlyA, actA and iap. The L. monocytogenes isolates were further subjected for multiplex-PCR-based serotyping. From 1002 samples screened, 16 (1.60%) were found positive for Listeria spp. Of these, seven samples (0.7%) were confirmed as L. monocytogenes and nine (0.9%) as L. innocua. All the seven isolates of L. monocytogenes were haemolytic, CAMP-positive, PI-PLC-positive, hlyA, pclA and prfA-positive by PCR, while only four isolates turned out to be PC-PLC-positive (opaque zone surrounding the growth). All the seven isolates of L. monocytogenes were serotyped as 4b. In conclusion, the PI-PLC assay and the virulence genes targeted PCR (plcA, prfA and hlyA plcA, prfA and actA genes for L. monocytogenes) hold a promise for rapid and reliable in vitro alternatives to in vivo pathogenicity tests.