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Abstract Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and a valuable genetic resource for wheat improvement. Several reference-quality genome sequences have been reported for A. tauschii accession AL8/78. A new genome sequence assembly (Aet v6.0) built from long Pacific Biosciences HiFi reads and employing an optical genome map constructed with a new technology is reported here for this accession. The N50 contig length of 31.81 Mb greatly exceeded that of the previous AL8/78 genome sequence assembly (Aet v5.0). Of 1,254 super-scaffolds, 92, comprising 98% of the total super-scaffold length, were anchored on a high-resolution genetic map, and pseudomolecules were assembled. The number of gaps in the pseudomolecules was reduced from 52,910 in Aet v5.0 to 351 in Aet v6.0. Gene models were transferred from the Aet v5.0 assembly into the Aet v6.0 assembly. A total of 40,447 putative orthologous gene pairs were identified between the Aet v6.0 and Chinese Spring wheat IWGSC RefSer v2.1 D-subgenome pseudomolecules. Orthologous gene pairs were used to compare the structure of the A. tauschii and wheat D-subgenome pseudomolecules. A total of 223 structural differences were identified. They included 44 large differences in sequence orientation and 25 differences in sequence location. A technique for discriminating between assembly errors and real structural variation between closely related genomes is suggested.