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Starch, as the main component of grain in cereals, serves as the major source of calories in staple food and as a raw material for industry. As the technological and digestive properties of starch depend on its content, the management of its components, amylose and amylopectin, is of great importance. The starch properties of wheat grain can be attuned using allelic variations of genes, including granule-bound starch synthase I (GBSS I), or <i>Wx</i>. The tertiary gene pool, including wheatgrass (<i>Thinopyrum</i>) species, provides a wide spectrum of genes-orthologs that can be used to increase the allelic diversity of wheat genes by wide hybridization. Octaploid partial wheat–wheatgrass hybrids (amphidiploids, WWGHs) combine the complete genome of bread wheat (BBAADD), and a mixed genome from the chromosomes of intermediate wheatgrass (<i>Thinopyrum intermedium</i>, genomic composition J^rJ^rJ^vsJ^vsStSt) and tall wheatgrass (<i>Th. ponticum</i>, JJJJJJJ^sJ^sJ^sJ^s). Thus, WWGHs may carry <i>Wx</i> genes not only of wheat (<i>Wx-B1</i>, <i>Wx-A1</i> and <i>Wx-D1</i>) but also of wheatgrass origin. We aimed to assess the level of amylose in starch and investigate the polymorphism of <i>Wx</i> genes in 12 accessions of WWGHs. Additionally, we characterized orthologous <i>Wx</i> genes in the genomes of wild wheat-related species involved in the development of the studied WWGHs, <i>Th. intermedium</i> and <i>Th. ponticum</i>, as well as in the putative donors of their subgenomes, bessarabian wheatgrass (<i>Th. bessarabicum</i>, J^bJ^b) and bluebunch wheatgrass (<i>Pseudoroegneria stipifolia</i>, St₁St₁St₂St₂). Although no significant differences in amylose content were found between different WWGH accessions, SDS-PAGE demonstrated that at least two WWGHs have an additional band. We sequenced the <i>Wx</i> gene-orthologs in <i>Th. bessarabicum</i>, <i>P. stipifolia</i>, <i>Th. intermedium</i> and <i>Th. ponticum</i>, and developed a WXTH marker that can discriminate the <i>Thinopyrum Wx</i> gene in the wheat background, and localized it to the 7E chromosome in <i>Th. elongatum</i>. Using the WXTH marker we revealed the allelic polymorphism of the <i>Thinopyrum Wx</i> gene in the studied WWGHs. The applicability of <i>Thinopyrum Wx</i> genes in wheat breeding and their effect on starch quality are discussed.