The genus Listeria encompasses six bacterial species L. monocytogenes，L. ivanovii，L. seeligeri，L. innoc- ua，L. welshimeri and L. grayi. Although these species resemble each other morphologically，ecologically，biochemi­cally and genetically，they show varied pathogenic potential. L. monocyto genes is a significant human，animal and a­quatic pathogen with clinical manifestation ranging from mild flu - like symptoms and gastroenteritis to septicemia， central nervous infection and feto - maternal infection. Through comparative genomics analysis，we used the iap geneencoding invasion - associated protein common to all members of the genus Listeria as the target because the compari­son of all iap genes indicated there were conserved gene portions at the 5 ’ and 3 ’ ends，while the internal portions were species specific. In addition，the 1+22 - 228 gene region was specific in L. ivanovii. A novel multiplex PCR， based on +22 - 228 in combination with optimized iap migration profiles，was developed for simultaneous identifica­tion of Listeria species，with a detection limit of down to 10² CFU/mL. Other 15 bacterial species did not show any amplicons via this PCR reaction. Furthermore，this assay was evaluated by 135 suspected Listeria isolates from aquatic products and corrected 4 and 5 misidentifications by Listeria selective agar plate screening and API system resjDective- ly. Therefore，this one - step molecular assay provides a rapid，accurate，reliable and inexpensive screening test to detect and differentiate Listeria species particularly the pathogenic species in surveillance programs concerning aquatic products safety and foodbome disease cases.