Genome architecture reveals hidden strain-level diversity in the highly conserved fish pathogen Nocardia seriolae

Fish nocardiosis, caused by <i>Nocardia seriolae</i>, poses a persistent threat to the aquaculture industry. Yet, the genomic determinants underlying strain-level diversity and adaptation remain poorly understood due to the high nucleotide conservation of this species. The objective of this study was to characterize genome-level variation among all publicly available complete genomes using integrative comparative genomics approaches that extend beyond nucleotide identity metrics. Nine complete genomes were analysed using average nucleotide identity, whole-genome structural rearrangement analysis, single-copy phylogenomics, genomic island prediction, pangenome reconstruction, functional annotation and antimicrobial resistance and virulence profiling. Although average nucleotide identity values confirmed extreme nucleotide conservation across all strains, extensive strain-specific structural rearrangements, including inversions, translocations and duplications, were detected. Phylogenomic reconstruction resolved geographically associated lineages despite minimal nucleotide divergence. Pangenome analyses supported an open pangenome dominated by a large, conserved core genome with limited but persistent accessory gene content and core biased gene duplication. Functional profiling revealed enrichment of transcriptional and metabolic processes, while resistance and virulence analyses identified only conserved intrinsic determinants shared across all strains. These findings demonstrate that genome architecture and pangenome dynamics provide critical resolution for understanding <i>N. seriolae</i> diversification. The study highlights the importance of integrating structural genomics and phylogenomics for strain tracking and surveillance in aquaculture systems.