Listeria monocytogenes (L. monocytogenes) is a deadly foodborne pathogen affecting populations, although most foodborne pathogens cause considerable illness but little fatality. Raw milk and uncooked dairy products are frequently associated with notable Listeria attacks. Microbial virulence traits are potential markers for discriminating the invasive and environmental L. monocytogenes variants. So, this study explored the bacteriological and molecular aspects of L. monocytogenes isolates, their virulence genes (hlyA, inIB, and prfA), and antimicrobial sensitivity in raw milk in Assiut, Egypt. One hundred raw milk samples (40 Cow, 20 markets, 40 Buffalo) were analyzed. L. monocytogenes was detected in only 11 (27.5 %), 8 (40 %) and 6 (15 %) of collected samples from cow, market and buffalo raw milk, respectively. These isolates carried the virulence hlyA, InlB, and prfA genes and were sensitive to Sulfamethoxazole trimethoprim (SXT (and Ciprofloxacin (CIP) followed by Chloramphenicol (C), less sensitivity to Gentamicin (CN), and resistant to Erythromycin (E) and Amoxicillin (AX).