Identification of novel Flavobacterium species from fish samples has growing importance due to their notorious effects on freshwater fish species. During the course of studying farmed rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) samples exhibiting clinical signs in Rize, Turkey, two isolates were recovered, and their taxonomic positions were revealed based on a genome-based polyphasic taxonomy approach. Pairwise sequence analyses of the 16S rRNA genes confirmed that strains F-372T and F-408 belonged to the genus Flavobacterium. Strain F-372T exhibited the highest identity level of 98.37% with Flavobacterium terrigena DSM 17934T (=KCTC 12761T) while the 16S rRNA gene sequence identity between strain F-372T and F-408 was 99.8% A polyphasic taxonomy approach including phenotypic characterization as well as genomic and chemotaxonomic analyses was employed to confirm the novelty of the strains in the genus Flavobacterium. The major fatty acids were isoC15:0 and C15:1 omega 6c for both strains while their predominant menaquinone was MK-6. Both strains have genome size of 3.3 Mb. The genomic DNA G + C content of the strains was 30.89 mol%. In addition, strain F-372T was found to carry 68 high confidence (HC) functional genes encoding AMR and virulence factors implying a possible correlation between those factors and mortality in the rainbow trout. Phylogenomic analysis and whole genome nucleotide sequences confirmed that the isolates represent a new species for which the name Flavobacterium bernardetii sp. nov. is proposed, with F-372T as the type strain (=JCM 34204T = KCTC 82264 T).