Since 2013, spontaneous mortality events have occurred sporadically in catfish hatcheries in the Mississippi Delta. Vibrio spp. are consistently isolated from moribund and dead fish. Histological analysis of moribund fish indicate the presence of a generalized bacterial septicemia. Phenotypic testing using the BBL CrystalTM enteric/nonfermenter id kit and gyrB sequencing identified these Vibrio spp. as V. cholerae. To better understand the origin of these isolates, a comparative analysis was performed on 66 suspected Vibrio spp. from cooperating fish health laboratories in North America. Multi-locus sequence analysis (MLSA) targeting the pyrH, rpoA, and recA genes revealed multiple V. cholerae variants, along with other Vibrio spp., including V. vulnificus, V. mimicus, V. metoecus, and V. harveyi. Molecularly confirmed V. cholerae isolates were subjected to PCR screening for the cholera toxin, deeming all catfish isolates PCR negative for the ctxAB genes. Phylogenetic analysis from pyrH>recA>rpoA concatenates revealed isolates from catfish formed multiple heterologous groups, which were distinct from isolates from ornamental fish. Moreover, fish isolates clustered separate from representative O1/O139 V. cholerae strains, suggesting the isolates from catfish hatcheries and ornamental fish are non-toxigenic, non-O1/O139 V. cholerae. Repetitive sequence mediated PCR employing the GTG5, ERIC II, and ERIC I&II primer sets agreed with MLSA, revealing marked genetic diversity among catfish isolates. Disc- diffusion assays were used to assess isolate sensitivity to Aquaflor® [florfenicol], Terramycin® [oxytetracycline], and Romet® [5:1 sulfadimethoxine/ormetoprim]. All catfish isolates were responsive to the three tested antimicrobials, with inhibition zones >20 mm. Genome sequencing using Oxford Nanopore Technology was performed and assembly/analysis is ongoing. Comparative genomics will be used to better illuminate the relationship of these V. cholerae isolates to environmental strains and those associated with human and aquatic animal health. This work lays the foundation for future studies investigating the origins of V. cholerae outbreaks in catfish hatcheries in the catfish farming region of the Mississippi Delta.