While both virulent and putatively avirulentYersinia ruckeristrains exist in aquaculture environments, the relationship between the distribution of virulence-associated factors andde factopathogenicity in fish remains poorly understood. Pan-genome analysis of 18 complete genomes, representing established virulent and putatively avirulent lineages ofY.ruckeri, revealed the presence of a number of accessory genetic determinants. Further investigation of 68 draft genome assemblies revealed that the distribution of certain putative virulence factors correlated well with virulence and host-specificity. The inverse-autotransporter invasin locusyrIlmwas, however, the only gene present in all virulent strains, while absent in lineages regarded as avirulent. Strains known to be associated with significant mortalities in salmonid aquaculture display a combination of serotype O1-LPS andyrIlm, with the well-documented highly virulent lineages, represented by MLVA clonal complexes 1 and 2, displaying duplication of theyrIlmlocus. Duplication of theyrIlmlocus was further found to have evolved over time in clonal complex 1, where some modern, highly virulent isolates display up to three copies.