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The construction of the 28-chromosome karyotype of <i>Dasypyrum breviaristatum</i> was undertaken using multicolor non-denaturing fluorescent in situ hybridization (ND-FISH) and Oligo-FISH painting protocols. A novel wheat-<i>D. breviaristatum</i> line D2138 contained 44 chromosomes including a pair of <i>D. breviaristatum</i> 6V^bS.2V^bL translocation chromosomes. Individual F₂ and F₃ progenies of a cross between D2138 with wheat lines CM62, MY11 and JM22, respectively, were characterized using ND-FISH and molecular markers. A relatively high chromosome alteration rate within wheat and <i>D. breviaristatum</i> 6V^bS and 2V^bL was observed in the three progeny populations, suggesting that chromosome 6V^bS.2V^bL has a gametocidal-like gene. The different types of translocation and deletion lines allowed localization of <i>D. breviaristatum</i>-specific gliadin coding genes on sub-telomeric regions of 6V^bS by PCR and acid polyacrylamide gel electrophoresis analysis. The positive effect of the <i>D. breviaristatum</i> 6V^bS on agronomic and quality characters was also demonstrated. The new wheat-<i>D. breviaristatum</i> 6V^bS and 2V^bL translocation lines will be useful as novel germplasm for breeding purposes.