Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most common causes of bacterial foodborne infections worldwide. It has an extensive host range, including birds and humans, making it one of the most adaptable Salmonella serovars. This study aims to define the virulence gene profile of S. Enteritidis and identify genes critical to its host specificity. Currently, there is limited understanding of the molecular mechanisms that allow S. Enteritidis to continue as an important foodborne pathogen. To better understand the genes that may play a role in the host-specific virulence and/or fitness of S. Enteritidis, we first compiled a virulence gene profile-based genome analysis of sequenced S. Enteritidis strains isolated from shell eggs in our laboratory. This analysis was subsequently used to compare the representative genomes of Salmonella serovars with varying host ranges and S. Enteritidis genomes. The study involved a comprehensive and direct examination of the conservation of virulence and/or fitness factors, especially in a host-specific manner—an area that has not been previously explored. Key findings include the identification of 10 virulence-associated clusters of orthologous genes (COGs) specific to poultry-colonizing serovars and 12 virulence-associated COGs unique to human-colonizing serovars. Virulence/fitness-associated gene analysis identified more than 600 genes. The genome sequences of the two S. Enteritidis isolates were compared to those of the other serovars. Genome analysis revealed a core of 2817 COGs that were common to all the Salmonella serovars examined. Comparative genome analysis revealed that 10 virulence-associated COGs were specific to poultry-colonizing serovars, whereas 12 virulence-associated COGs were present in all human-colonizing serovars. Phylogenetic analyses further highlight the evolution of host specificity in S. Enteritidis. This study offers the first comprehensive analysis of genes that may be unique to and possibly essential for the colonization and/or pathogenesis of S. Enteritidis in various and specific hosts.