ABSTRACT: Recent reports suggest an emergence of novel Chryseobacterium spp. associated with aquaculture-reared fish worldwide. Herein, we report on multiple Chryseobacterium spp. infecting Great Lakes fishes that are highly similar to previously detected isolates from Europe, Africa, and Asia but have never before been reported in North America. Polyphasic characterization, which included extensive physiological, morphological, and biochemical analyses, fatty acid profiling, and phylogenetic analyses based upon partial 16S rRNA gene sequences, highlighted the diversity of Great Lakes’ fish-associated chryseobacteria and also suggested that at least 2 taxa represent potentially novel Chryseobacterium spp. Screening for the ability of representative chryseobacteria to elicit lesions in experimentally challenged fish showed that they induced varying degrees of pathology, some of which were severe and resulted in host death. Median lethal dose (LD₅₀) experiments for the isolate that elicited the most extensive pathology (Chryseobacterium sp. T28) demonstrated that the LD₅₀ exceeded 4.5 × 10⁸ cfu, thereby suggesting its role as a facultative fish-pathogenic bacterium. Histopathological changes in T28-infected fish included epithelial hyperplasia of the secondary lamellae and interlamellar space that resulted in secondary lamellar fusion, monocytic infiltrate, and mucus cell hyperplasia, all of which are consistent with branchitis, along with monocytic myositis, hemorrhage within the muscle, liver, adipose tissue, and ovaries, spongiosis of white matter of the brain, multifocal edema within the granular cell layer of the cerebellar cortex, and renal tubular degeneration and necrosis. The findings of this study underscore the widespread presence of chryseobacteria infecting Great Lakes fish.