Brucellosis has a wide range of clinical severity in humans that remains poorly understood. Whole genome sequencing (WGS) analysis may be able to detect variation in virulence genes. We used Brucella melitensis sequences in the NCBI Sequence Read Archive (SRA) database to assemble 248 whole genomes, and additionally, assembled 27 B. melitensis genomes from samples of human patients in Southern Israel. We searched the 275 assembled genomes for the 43 B. melitensis virulence genes in the Virulence Factors of Pathogenic Bacteria Database (VFDB) and 10 other published putative virulence genes. We explored pan-genome variation across the genomes and in a pilot analysis, explored single nucleotide polymorphism (SNP) variation among the ten putative virulence genes. More than 99% of the genomes had sequences for all Brucella melitensis virulence genes included in the VFDB. The 10 other virulence genes of interest were present across all the genomes, but three of these genes had SNP variation associated with particular Brucella melitensis genotypes. SNP variation was also seen within the Israeli genomes obtained from a small geographic region. While the Brucella genome is highly conserved, this novel and large whole genome study of Brucella demonstrates the ability of whole genome and pan-genome analysis to screen multiple genomes and identify SNP variation in both known and novel virulence genes that could be associated with differential disease virulence. Further development of whole genome techniques and linkage with clinical metadata on disease outcomes could shed light on whether such variation in the Brucella genome plays a role in pathogenesis.