This study aimed to evaluate the hazards posed by foodborne bacteria of the Listeria genus by analyzing prevalence, diversity and virulence of Listeria spp. in food and food manufacturing plants. Seventy five isolates obtained from the routine analysis of 653 samples by three diagnostic laboratories in Northern Italy were genotipically differentiated by Repetitive Extragenic Palindrome (rep) PCR with the GTG5 primer, identified by sequencing the 16S rRNA gene and examined by specific PCR tests for the presence of L. monocytogenes virulence determinants occasionally found to occur in other species of the genus. The identity of the amplification products was confirmed by sequencing. Fifty seven isolates were identified as L. innocua, 12 as L. monocytogenes, 5 as L. welshimeri and one as L. seeligeri. All L. monocytogenes isolates belonged to the serotype 1/2a and were predicted to be virulent for the presence of the inlJ internalin gene. Potentially virulent strains of L. innocua, L. seeligeri and L. welshimeri, carrying the L. monocytogenes inlA gene and/or hly gene, were identified, and most isolates were found to possess the toxin-antitoxin system mazEF for efficient adaptation to heat shock. Results indicated the need to reinforce food contamination prevention measures against all Listeria species by efficiently defining their environmental distribution.