Genome constitution and genetic relationships between six Elymus species were assessed by physical mapping of different repetitive sequences using a technique of sequential fluorescence in situ hybridization and genomic in situ hybridization. The six Elymus species are all naturally growing species in northwest China, namely, E. sibiricus, E. nutans, E. barystachyus, E. xiningensis, E. excelsus, and E. dahuricus. An StStHH genome constitution was revealed for E. sibiricus and StStHHYY for the remainder species. Each chromosome could be clearly characterized by physical mapping with 18S-26S rDNA, 5S rDNA, Afa-family, and AAG repeats, and be allocated to a certain genome by genomic in situ hybridization. Two 5S rDNA sites, each in the H and St genomes, and three 18S-26S rDNA sites, two in the St genome and one in the Y genome, were uncovered in most of the species. The strong Afa-family hybridization signals discriminated the H genome from the St and Y genomes. The H and Y genome carried more AAG repeats than St. A common non-Robertsonian reciprocal translocation between the H and Y genomes was revealed in E. barystachyus, E. xiningensis, E. excelsus and E. dahuricus. Comparison of molecular karyotypes strongly suggests that they can be classified into three groups, namely, E. sibiricus, E. nutans, and others.