Walnut (Juglans) species are used as nut crops worldwide. Eastern black walnut (EBW, Juglans nigra), a diploid, horticultural important woody species is native to much of eastern North America. Although it is highly valued for its wood and nut, there are few resources for understanding EBW genetics. Here, we present a high-quality genome assembly of J. nigra based on Illumina, Pacbio, and Hi-C technologies. The genome size was 540.8 Mb, with a scaffold N50 size of 35.1 Mb, and 99.0% of the assembly was anchored to 16 chromosomes. Using this genome as a reference, the resequencing of 74 accessions revealed the effective population size of J. nigra declined during the glacial maximum. A single whole-genome duplication event was identified in the J. nigra genome. Large syntenic blocks among J. nigra, Juglans regia, and Juglans microcarpa predominated, but inversions of more than 600 kb were identified. By comparing the EBW genome with those of J. regia and J. microcarpa, we detected InDel sizes of 34.9 Mb in J. regia and 18.3 Mb in J. microcarpa, respectively. Transcriptomic analysis of differentially expressed genes identified five presumed NBS-LRR (NUCLEOTIDE BINDING SITE-LEUCINE-RICH REPEAT) genes were upregulated during the development of walnut husks and shells compared to developing embryos. We also identified candidate genes with essential roles in seed oil synthesis, including FAD (FATTY ACID DESATURASE) and OLE (OLEOSIN). Our work advances the understanding of fatty acid bioaccumulation and disease resistance in nut crops, and also provides an essential resource for conducting genomics-enabled breeding in walnut.

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