A first sequence of the Ae. speltoides B chromosome has been available since 2020 (RUBAN et al. 2020b), but this assembly represents ~16% of the B only and thus cannot be used to uncover B-encoded genes. Now, high-molecular-weight DNA from +B leaf tissue of the clonally propagated +B plant was used to generate a chromosome-scale genome assembly. A total of 154 Gb of PacBio HiFi reads and 36.4 Gb of Nanopore reads (>25 kb) were generated for primary assembly using hifiasm (Cheng et al. 2026). The resulting 5.47 Gb assembly achieved 93.7% BUSCO completeness (contig N50 = 17.1 Mb). Approximately 104 Gb of Hi-C sequencing data derived from leaf tissue of the same +B plant were employed to scaffold the primary contigs. The assembly yielded eight large scaffolds (398–835 Mb), each displaying a characteristic Rabl configuration. Alignment of these scaffolds to the reference genome of Ae. speltoides accession AEG-9674-1 without B chromosome (Avni et al. 2022) revealed that seven of the eight scaffolds showed strong synteny with the standard A chromosomes 1S–7S. To determine whether the remaining large scaffold corresponded to the B chromosome, we generated ~60 Gb of whole-genome sequencing (WGS) data from 0B AR-derived lateral root tissue of the same plant, as well as approximately 36 Gb of WGS data from +B leaf tissue. Comparative read-mapping analyses showed that the eighth scaffold exhibited normal sequencing coverage in +B leaf-derived data but substantially reduced coverage in 0B AR-derived data. Thus, the 398 Mb scaffold represents the B chromosome, accounting for 69% of its size as estimated by flow cytometry. Additionally, 4.81 Gb of contigs were assigned to the seven pairs of A chromosomes, representing 91% of their estimated size (1C=5.27 Gb). Consequently, we produced a high-quality chromosome-scale assembly of Ae. speltoides carrying B chromosomes. To identify genes associated with the B chromosome elimination process, RNA-seq was performed across developmental stages and different tissues in which B chromosome behavior differs. Using all +B RNA-seq datasets, we annotated the Ae. speltoides genome assembly containing the B chromosome. This annotation identified 59,981 transcripts and 47792 protein-coding genes on the seven A chromosomes and 5,940 transcripts and 4,196 protein-coding genes on the B chromosome.