Two yellow-pigmented isolates, F-60(T) and F-392, were isolated from the internal organs of an apparently healthy rainbow trout (Oncorhynchus mykiss). The strains were identified as members of the genus Flavobacterium based on the results of 16S rRNA gene sequence analysis. Strains F-60(T) and F-392 had the highest 16S rRNA gene sequence identity level of 97.4% to the type strain of Flavobacterium crassostreae LPB0076(T). A polyphasic taxonomic approach including phenotypic, chemotaxonomic and genomic characterization was employed to ascertain the taxonomic position of the strains within the genus Flavobacterium. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity based on BLAST (ANIb) values for strains F-60(T) and F-392 were calculated as 100%. However, dDDH and ANI analyses between the strains and their close neighbours confirmed that both strains represent a novel species in the genus Flavobacterium. The strains shared the highest dDDH and ANIb levels of 23.3 and 77.9%, respectively, with the type strain of Flavobacterium frigidarium DSM 17623(T) while those values for F crassostreae LPB0076(T) were obtained as 21.4-21.5% and 76.3%. The DNA G+C content of the strains was 34.5 mol%. Chemotaxonomic and phylogenomic analyses of these isolates confirmed that both strains are representatives of a novel species for which the name Flavobacterium muglaense sp. nov. is proposed, with F-60(T) as the type strain (=JCM 34196(T) =KCIC 82256(T)).