The genome constitution of Elymus dahuricus and E. nutans were characterized using 12 repetitive sequence by a technique of fluorescence in situ hybridization (FISH) probes, which include probes of nine trinucleotide simple sequence repeat (SSR) motifs and two satellite DNA repeats sequences pAs1 and pSc119.2, and ribosomal 5S rDNA. The results suggested that: (1) All sequences produce detectable hybridization signals on the chromosomes of both species. In both species, (AAC)10, (ACT)10, and (CAT)10 were revealed to be co-localized, whereas AAG and AGG also revealed an approximately co-localized. All repetitive sequences except 5S rDNA, produced high-intensity and multiple hybridization sites on the H chromosomes. The hybridization sites of different repeat sequence on the both St and Y chromosomes are varied. It showed that 5S rDNA, pSc119.2, (AAC) 10, (CAT) 10, (ACT) 10, (CAC) 10 have fewer or no signal distribution, while the remainders have more signal sites. (2) E. dahuricus included a hybridization site of (AAC)10, (ACT)10, (CAT)10, (CAC)10 in St genome. It is distinct from E. nutans which includes none of them in St genome. E. hahuricus has one unique pSc119.2 hybridization in the terminal position of a pair of chromosome, in contrast to the E. nutans involving it in the intercalary region of a pair chromosome in St genome. E. nutans contains more repetitive sequences hybridization site in St and Y genome than E. dahuricus. (3) A unique H/Y inter-genomic translocation was stably detected between different materials in E. dahuricus. It implies a relative genome stability of E. dahuricus. However, more polymorphic hybridization sites in H genome than in St and Y genome was still observed in E.dahuricus between different materials. E. nutans present higher number of polymorphic hybridization sites between different materials than E. dahuricus. According to the study, both H genomes of E. nutans and E. dahuricus are derived from H. bogdanii, and St genome may be originated from different species of Pseudoroegneria. Compared with E. dahuricus, St and Y genomes of E. nutans may have higher chromosomal structural variability, and the reason for the large variation of St and Y genomes of E. nutans may be the introgression hybridizations with the species which contains StY genome distributed in the same region. This study provides a new perspective and evidence for the speciation and evolution of E. nutans and E. dahuricus at the level of molecular cytogenetics.