Two strains (T16(T) and S12) of a Gram-reaction-negative, rod-shaped, yellow-pigmented bacterium were recovered from kidneys of feral spawning adult Chinook salmon (Oncorhynchus tshawytscha) and the gills of captive-reared Chinook salmon fingerlings suffering a mortality episode, respectively. Polyphasic characterization revealed that these strains possessed gliding motility, contained a flexirubin-type pigment, proteolysed multiple substrates (i.e. gelatin, casein and elastin), and had a mean DNA G+C content of 35.6 mol%. Sequencing of the 16S rRNA gene demonstrated that strains T16(T) and S12 were nearly identical to one another (>99% similarity) and were placed within the genus Flavobacterium, with Flavobacterium aquidurense CCUG 59847(T) (98.3%), Flavobaterium araucananum CCUG 61031(T) (98.2%) and Flavobacterium frigidimaris (AB183888, 98.1%) being their closest relatives. Subsequent phylogenetic analyses using neighbour-joining, maximum-parsimony and Bayesian methodologies demonstrated that strains T16(T) and S12 formed a well-supported clade within the genus Flavobacterium that was distinct from other Flavobacterium species. The major fatty acid constituents of strains T16(T) and S12 were iso-C15 : 0, C16 : 1ω6c and/or C16 : 1ω7c, iso-C17 : 0 3-OH, and iso-C15 : 0 3-OH according to fatty acid methyl ester analysis. The mean level of DNA-DNA relatedness between strain T16(T) and F. aquidurense CCUG 59847(T) and F. araucananum CCUG 61031(T) was 23 and 29%, respectively. Thus, the data accumulated in this study support the suggestion that strains T16(T) and S12 represent a novel species of the genus Flavobacterium, for which the name Flavobacterium spartansii sp. nov. is proposed. The type strain is T16(T) ( = LMG 27337(T) = ATCC BAA-2541(T)). Emended descriptions of F. aquidurense and F. araucananum are also proposed.