The perennial species Dasypyrum breviaristatum (genome Vb) contains many potentially valuable genes for the improvement of common wheat. Construction of a detailed karyotype of D. breviaristatum chromosomes will be useful for the detection of Dasypyrum chromatin in wheat background. We established the standard karyotype of 1Vb-7Vb chromosomes through nondenaturing fluorescence in situ hybridization (ND-FISH) technique using 28 oligonucleotide probes from the wheat - D. breviaristatum partial amphiploid TDH-2 (AABBVbVb) and newly identified wheat - D. breviaristatum disomic translocation and addition lines D2138 (6VbS.2VbL), D2547 (4Vb), and D2532 (3VbS.6VbL) by comparative molecular marker analysis. The ND-FISH with multiple oligo probes was conducted on the durum wheat - D. villosum amphiploid TDV-1 and large karyotype differences between D. breviaristatum and D. villosum was revealed. These ND-FISH probes will be valuable for screening the wheat - Dasypyrum derivative lines for chromosome identification, and the newly developed wheat - D. breviaristatum addition lines may broaden the gene pool of wheat breeding. The differences between D. villosum and D. breviaristatum chromosomes revealed by ND-FISH will help us understand evolutionary divergence of repetitive sequences within the genus Dasypyrum.