Bacterial pathogens often show significant intra-species variations in ecological fitness, host preference and pathogenic potential to cause infectious disease. The species of Listeria monocytogenes, a facultative intracellular pathogen and the causative agent of human listeriosis, consists of at least three distinct genetic lineages. Two of these lineages predominantly cause human sporadic and epidemic infections, whereas the third lineage has never been implicated in human disease outbreaks despite its overall conservation of many known virulence factors. The genomes of 26 L. monocytogenes strains representing the three lineages are compared based on both in silico comparative genomic analysis and high-density, pan-genomic DNA microarray hybridizations. We uncover 86 genes and 8 small regulatory RNAs that likely make L. monocytogenes lineages differ in carbohydrate utilization and stress resistance during their residence in natural habitats and passage through the host gastrointestinal tract. We also identify 2,330 to 2,456 core genes that define this species along with an open pan-genome pool that contains more than 4,052 genes. Phylogenomic reconstructions based on 3,560 homologous groups allowed robust estimation of phylogenetic relatedness among L. monocytogenes strains. The pan-genome approach enables accurate co-analysis of DNA sequence and hybridization array data for both core gene estimation and phylogenomic reconstruction. Application of our method to the pan-genome of L. monocytogenes sheds new insights into the intra-species genomic diversification, niche expansion and evolution of this important foodborne pathogen.