In this article, we developed a biomolecular assay specifically designed for the identification of Listeria at species level. The proposed test was based on (i) a duplex PCR targeting a specific fragment for L. monocytogenes (plcA gene) and a common fragment for all the Listeria (16S rRNA gene) and (ii) a minisequencing of the common fragment to detect diagnostic sites for the differentiation of the other five species: L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, and L. grayi. The specificity of the assay was first tested on a total of 25 certified strains representing 6 Listeria species and 14 non-Listeria strains as negative control and validated on 124 wild isolates obtained from food samples. The proposed assay provides an appropriate tool for rapid identification of Listeria at species level and it should be of great benefit to the food industry as well as to regulatory or public health laboratories engaged in establishing the safety of food products and the management of listeriosis.