S. Pullorum and S. Enteritidis are closely related in genetic terms, but they show very different pathogenicity and host range. S. Enteritidis infects many different hosts, usually causing acute gastroenteritis, while S. Pullorum is restricted to avian, where it causes systemic disease in young animals. The reason why they differ in host range and pathogenicity is unknown. The core-genome denotes those genes that are present in all strains within a clade, and in the present work, an automated bioinformatics workflow was developed and applied to identify core-genome differences between these two serovars with the aim to identify genome features associated with host specificity of S. Pullorum. Results showed that S. Pullorum unique coding sequences (CDS) were mainly concentrated in three regions not present in S. Enteritidis, suggesting that such CDS were taken up probably during the separation of the two types from their common ancestor. One of the unique regions encoded Pathogenicity Islands 19 (SPI-19), which encodes a type VI secretion system (T6SS). Single-nucleotide polymorphism (SNP) analysis identified 1791 conserved SNPs in coding sequences between the two serovars, including several SNPs located in a type IV secretion system (T4SS). Analyzing of 100 bp regions upstream of coding sequences identified 443 conserved SNPs between the two serovars, including SNP variations in type III secretion system effector (T3SE). In conclusion, this analysis has identified genetic features encoding putative factors controlling host-specificity in S. Pullorum. The novel bioinformatic workflow and associated scripts can directly be applied to other bacteria to uncover the genome difference between clades.