Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish. Four discrete genetic groups exist within the species and research has demonstrated associated host and virulence differences . Previous research has suggested that the species designation may require revisions; therefore, t he present study determined the taxonomic status of the four genetic groups of F. columnare using polyphasic and phylogenomic approaches . A polyphasic approach was taken to confirm previous phylogenetic relationships and to compare phenotypic, biochemical, and chemotaxonomic properties or representative isolates from the four genetic groups. The research followed the proposed minimal standards for describing new taxa of the family Flavobacteriaceae by Bernardet et al. Phylogenetic analyses of 16S rRNA and gyrB genes using different methodologies demonstrated the four genetic groups formed well-supported and distinct clades within the genus Flavobacterium . The average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) values between F. columnare ATCC 23463T, genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT were less than 90.84% and 42.7%, respectively. Chemotaxonomic, MALDI-TOF characterization and ANI/GGDC calculations afforded differentiation between the genetic groups, indicating each group is a discrete species. T he names F. covae sp. nov. , F. davisii sp. nov. , and F. oreochromis sp. nov. were proposed to represent genetic groups 2, 3, and 4, respectively, and recently validated. Since these pathogens (collectively referred to as columnaris causing bacteria, CCB) are globally distributed and have significant impacts on wild and cultured fish species, recognition of the four species will advance and improve research to define host-pathogen-environment relationships, epidemiology, and develop effective control and prevention measures in aquaculture. Such research needs to target the correct bacterial species and research findings can be properly interpreted by correct and consistent taxonomic assignment.