Abstract: This study aimed to characterize the biological properties, pathogenicity, antimicrobial resistance profile, and whole-genome features of Yersinia ruckeri, a potential pathogen associated with overwintering syndrome in grass carp (Ctenopharyngodon idella), to provide a scientific basis for etiological understanding and disease management. A predominant bacterial strain, Ci2537, was isolated from the liver tissue of diseased grass carp. Morphological observation, Gram staining, 16S rRNA gene sequencing, and phylogenetic analysis confirmed its identity as Y. ruckeri, exhibiting short rod-shaped morphology with peritrichous flagella under transmission electron microscopy. Multilocus sequence typing (MLST) assigned the strain to sequence type ST-32 of biotype I. Artificial infection challenge verified its pathogenicity, inducing clinical signs including cutaneous hemorrhage, intestinal congestion and swelling, and hepatic hemorrhage in grass carp, with a median lethal dose (LD₅₀) of 1.1 × 10⁶ CFU/mL. Antimicrobial susceptibility testing via Kirby-Bauer disk diffusion and broth microdilution methods revealed susceptibility to eight antibiotics (e.g., trimethoprim-sulfamethoxazole, tetracycline, florfenicol) and resistance to four antibiotics (e.g., amoxicillin, penicillin). Whole-genome sequencing identified a circular chromosome of 3,727,672 bp (GC content: 47.29%) and a plasmid of 143,069 bp (GC content: 39.87%), encoding 3,446 predicted coding sequences (CDS). Genomic annotation detected eight antibiotic resistance genes and multiple virulence-associated factors. Comparative genomic analysis indicated that strain Ci2537shares the closest phylogenetic relationship with Y. ruckeri strain SC09 isolated from channel catfish (Ictalurus punctatus). In conclusion, the grass carp-derived Y. ruckeri strain Ci2537 (ST-32) demonstrates high pathogenicity, harbors diverse virulence and resistance determinants, and exhibits close evolutionary affinity with a channel catfish-origin strain. This study provides critical genomic and phenotypic evidence supporting the role of Y. ruckeri in grass carp overwintering syndrome and offers a foundation for targeted antimicrobial therapy, vaccine development, and evidence-based prevention strategies in aquaculture.