Salmonella enterica subspecies enterica serovar Gallinarum biovar Pullorum (Salmonella Pullorum) has strict host specificity for poultry, and pullorum disease seriously threatens the poultry industry. Virulence genes play a central role in Salmonella pathogenicity, but very few reports are available on the distribution of virulence genes in Salmonella Pullorum. In this study, we investigated 304 Salmonella Pullorum isolates recovered from chickens in China between 1953 and 2015 for the presence of 25 Salmonella virulence genes (invA, orgA, prgH, sitC, spaN, sifA, spiA, ttrC, mgtB, misL, siiE, spi4D, pipA, sipB, sopB, sefA, cdtB, pagC, shdA, msgA, lpfC, tolC, iroN, pefA, and spvB), including pathogenicity island genes, fimbriae genes, and virulence plasmid genes. PCR showed that 15 of the 25 virulence genes were present in all isolates tested, whereas cdtB was not present in any isolate. The presence rates of the remaining genes ranged from 97.7% to 99.7%. The variation rates of these virulence genes was low, and no significant differences were identified in the distribution of virulence genes over time. On the basis of the distribution of these virulence genes, the 304 Salmonella Pullorum isolates were divided into 10 virulence genotypes. The major genotype, which comprised 93.4% of all isolates, included isolates that carried 24 of the virulence genes assessed. The results of this study will help in the characterization of Salmonella Pullorum and in the study of the correlation between virulence genotypes and pathogenicity.