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Characterizing the nature of genetic differentiation among individuals and populations and its distribution across the genome is increasingly important to inform both conservation and management of exploited species. Atlantic Halibut (Hippoglossus hippoglossus) is an ecologically and commercially important fish species, yet knowledge of population structure and genomic diversity in this species remains lacking. Here, we use restriction-site associated DNA sequencing and a chromosome-level genome assembly to identify over 86 000 single nucleotide polymorphisms mapped to 24 chromosome-sized scaffolds, genotyped in 734 individuals across the Northwest Atlantic. We describe subtle but significant genome-wide regional structuring between the Gulf of St. Lawrence and adjacent Atlantic continental shelf. However, the majority of genetic divergence is associated with a large putative chromosomal …