Listeria monocytogenes is a foodborne pathogen, and one of most important zoonotic bacterium with fatality rates up to 20- 30%. Which has a great hazard on public health and dairy industry. Milk and dairy products are implicated in most outbreaks of listeriosis all over the world. This work was aimed to investigate the prevalence of Listeria species in milk, feces of dairy and pregnant women in two Provinces (Sharkia and Dakahlia) in Egypt. Also, to molecularly identify L. monocytogenes to the species level based on 16S rRNA gene and to determine the prevalence of some virulence genes (inlA and inlB) in molecularly identified L. monocytogenes isolates. Furthermore, to determine the genetic diversity of L. monocytogenes isolated from different sources by Eric-PCR. In this study, bacteriological analysis of 350 different samples including; normal raw milk (n=200), mastitis milk (n=50), feces of dairy cattle (n=50) and stool of pregnant women (n=50. Listeria spp. were isolated on Oxford agar and then subjected to biochemical and molecular identification. The results showed that: - Out of 350 examined samples, 56 (16%) were contaminated with Listeria spp. by using culture techniques, and biochemical tests. Among the 56 isolated Listeria spp. 38 (19%), 6 (12%), 8 (16%) and 4(8%) were recovered from normal raw milk, mastitis milk, feces of dairy cows and stool of pregnant women, respectively. The Listeria spp. isolates from different sources involved L. monocytogenes (4.28%), L. ivanovii (5%), L. grayi (2.5%), L. welshimeri (2.5%) and L. innocua (1%). - L. ivanovii was isolated from normal raw milk and mastitis milk (6%, each). The prevalence rates of L. ivanovii were 4% and 2% in feces of dairy cow samples and stool of pregnant women samples, respectively. -The prevalence rates of L. monocytogenes represented as 8% from feces of dairy cows followed by 4% from both normal raw milk and stool of pregnant women samples then 2% in mastitis milk samples. - The prevalence rates of L. welshimeri were 4% and 2% in normal raw milk and stool of pregnant women, respectively. L. welshimeri was not isolated from mastitis milk and feces of dairy cow. - L. grayi was isolated from 4%, 3% and 2% of feces of dairy cows, normal raw milk and mastitis milk samples, respectively. - L. innocua was isolated from normal raw milk and mastitis milk samples (2%, each). while it was not recovered from fecal samples of dairy cows and pregnant women. -In this study, the 16S rRNA gene was amplified in all examined (100%) L. monocytogenes isolates with an amplicon size of 1200 bp. - PCR was successfully amplified inlA and inlB genes at specific base pair 800 and 343, respectively. InlA gene was molecularly detected in 12 L. monocytogenes isolates (80%); (7/8) of normal raw milk, (1/1) of mastitis milk, (3/4) of feces of dairy cows and (1/2) of stool of pregnant women. However, inlB gene was observed in only 6 L. monocytogenes isolates (40%); (3/8) of normal raw milk, (1/1) of mastitis milk, (1/4) of feces of dairy cows and (1/2) of stool of pregnant women. -Moreover, two L. monocytogenes isolates from normal raw milk and feces of dairy cows didn’t harbor both inlA and inlB genes. -Eric-PCR is highly reproducible, discriminatory and effective molecular typing technique that plays a more important role in tracking and epidemiological investigations of L. monocytogenes bacteria by its ability to discerning closely related L. monocytogenes strains to confirm the sources of outbreaks, detect the reservoirs of epidemic strains and also to establish mode of transmission. -Eric-PCR of genomic DNA among 15 L. monocytogenes isolates from normal milk, mastitis milk and feces of dairy cows and stool of pregnant women yielded 11 DNA fingerprint profiles (namely R1-R11) with amplified fragments ranged between 100 and 3000 bp. The dendrogram clearly showed that 7 isolates of L. monocytogenes were divided into three clusters designated as (I, II & III); whereas the remaining were represented by 8 single isolates. -In cluster I, the similarity between L. monocytogenes isolates recovered from normal milk (LMM1) and feces of dairy cow (LMF1) was 83.3%, while between normal milk (LMM1) and stool of pregnant women (LMPW1) was 66.7%. However, the genentic similarity between feces of dairy cow (LMF1) and stool of pregenant women (LMPW1) isoletes was 57.1%. -In cluster II showed 50% similarity between two L. monocytogenes isolates (LMM3 & LMM4) recovered from normal milk of dairy cow. -In cluster III the similarity between two L. monocytogenes isolates from normal milk of dairy cow and stool of pregnant women (LMM6 & LMPW2) was 50%. -Eight fingerprint profiles (R4-R11) were generated and most L. monocytogenes isolates from different sources were found