This study was conducted to determine the prevalence of Listeria spp < em>. in milk and feces of dairy cows as well as stool of pregnant women in Sharkia and Dakahlia Governorates, Egypt. Three hundred and fifty samples including; normal raw milk (n=200), mastitis milk (n=50), feces of dairy cattle (n=50) and stool of pregnant women (n=50) were randomly collected. Listeria spp. were isolated on Oxford agar and then subjected to biochemical and molecular identification. The overall isolation rate of Listeria spp. was 16%. The prevalence rates of Listeria monocytogenes were 8%, 4%, 2% and 4% in feces of dairy cows, normal milk, mastitis milk and stool of pregnant women, respectively. The prevalence of L. ivanovii and L. welshimeri in milk of dairy cows were 6% and 4%, respectively. L. monocytogenes isolates were molecularly confirmed to the species level by amplification of 16S rRNA gene. The distribution of internalin A (inlA) and internalin B (inlB) was determined in 15 identified L. monocytogenes isolates. The overall prevalence of inlA gene and inlB gene in L. monocytogenes isolates from different sources were 80% and 40%, respectively. Two L. monocytogenes isolates from normal raw milk and feces of dairy cows didn't harbor both inlA and inlB genes. Listeria spp.  isolated from normal milk, feces, mastitis milk and stool of pregnant women, potentially can cause human illness and abortion in pregnant women. Internalin (A&B) genes are considered the best indicator for virulence determination of L. monocytogenes isolated from different sources.