Flavobacterium oreochromis, F. covae, F. davisii and F. columnare, columnaris-causing bacteria (CCBs) in freshwater fish, are long Gram-negative bacilli and with fastidious growth. Species of this genera affect several species of fish, both in natural populations and in productive environments. The species mentioned were recently classified by bringing together isolates from each of the 4 genetic groups (GGs) of the old delineation for the species F. columnare, which, due to its wide distribution and genetic diversity leads reclassification. With this new proposition, it will be necessary to carry out genetic of circulating strains to better understand the species involved in fish clinical cases. Strains of this study were stocked in the Laboratory of Microbiology and Parasitology of Aquatic Organisms at the Aquaculture Center of Unesp. Fishes from 6 species (Oreochromis niloticus, Piaractus mesopotamicus, Colossoma macropomum, Hypostomus sp., Pseudoplatystoma punctifer and Astyanax altiparanae) with signals columnaris-causing bacteria (CCBs) were sampled as cutaneous lesions with depigmented area, desquamation, gills congestion and cutaneous hemorrhage leading to sepsis. Of this clinical cases, 11 strains were isolated under aseptic laboratory protocols by plating the musculature and cranial kidneys and 1 strain was isolated from water of recirculation system. All strains of the study were isolated in G Agar plates cultivated at 28 ºC for 24-48 hours except for environmental strain that was isolated in G Agar supplemented with tobramycin at 1ug x mL-1. To the first trial suspected orange-yellow pigmented colonies were cultivated in G Agar and BHI agar. After colonies do not growth in conventional media, they were investigated for casein hydrolyze in G Agar supplemented with 2% of skimmed milk. As result all strains fulfill this two-method forming a “halo” of hydrolyzes in modified G Agar. Gliding motilities were observed in 9/11 of strains by the spreading. The unique isolates that do not express gliding in studied conditions were F. indicum and C. gambriani. Further the total genomic DNA of isolates were obtained using Blood and Tissue kit (Qiagen) according to manufactures recommendations. The DNA were analyzed by concentration and purity. Polymerase Chain Reactions were carried with Platinum II Hot Start Taq DNA polymerase (Invitrogen) the primers pair used to amplify the 1300pb 16S rRNA of Flavobacterium spp. was UN-20 and R1387, the amplified DNA were sent to sequencing procedure by Sange dideoxy nucleotide sequencing. The consensus sequence was built by paired end strategy, gene sequences were searched by BLAST (NCBI). As result the 11 strains was assigned into 5 species Flavobacterium oreochromis, F. davisii, F. indicum, F. inkyongense and Chryseobacterium gambriani, with >99% of similarity with respective RefSeq genomes by comparison of 1000-900 bp fragments. The water source isolate 16S rRNA sequence, show similarity of 99,88% with unidentified Flavobacterium sp. strains. The three top hits refer to strains isolated from gut of 2 species of fish and one strain isolated from ornamental aquaria. The results demonstrated that atypical species could be involved in diseases of freshwater fishes in Brazil and some isolates were found in coinfected organs, presupposing of these pathogens could be involved as primary or secondary agents of the diseases in fish.