Pan-genome analysis identified more and more genome structural variations with significant effects on phenotype and environmental adaptation in many plant species. However, in large and complex genomes such as wheat and its relatives, it’s difficult to reveal the chromosome-level variation and its effect based on the current sequencing and assembly techniques. Oligonucleotide probe multiplex (ONPM) provides a simple and efficient tool for identifying large-segmental chromosome variation in wheat and its relatives via fluorescence in situ hybridization (FISH). ONPM#4 containing eight oligonucleotides derived from three repeats (pAs1, pSc119.2, (GAA)10) developed in Nanjing Agricultural University, China, has been successfully used for chromosome identification in wheat and Dasypyrum villosum. Based on 184 signal variations in 662 modern wheat cultivars (lines), and 94 signal variations in 260 plants of 13 Dasypyrum villosum accessions, pan-karyotypes of the two species have been developed. In wheat, 37 core and 147 dispensable heterochromatin patterns were observed which produced 193 polymorphic chromosomes. In Dasypyrum villosum, 13 core and 81 dispensable patterns were seen which produced 106 polymorphic chromosomes. The two pan-karyotypes and dispensable heterochromatin blocks provide references for distinguishing more structural chromosome variations (SCVs) and genetic markers for population structure and genome-wide association analysis in order to reveal the effects of SCVs on phenotype and genome evolution of wheat and Dasypyrum villosum.