Elymus nutans and Elymus sibiricus, common species of Elymus genus of Triticeae tribe, are both tall-growing, perennial bunchgrass with drooping inflorescence. When collecting wild germplasm in field survey, there are some difficulties in rapid and accurate identification of the two similar species due to some overlapped morphological characters such as dense spikelets in some E. Sibiricus collections from upper elevations. We used SSR primers from different sources to characterize genetic variance on a total of 18 accession of E. nutans and E. sibiricus. The 12 selected primers produced 152 well defined bands, and the percentage of polymorphism was 88.6%. Two main clusters corresponded exactly to two species are shown in an arithmetic average (UPGMA) dendrogram, which is in agreement with the results of principal coordinate analysis (PCoA) analysis. The analysis of molecular variance (AMOVA) and the t-test of Dice coefficients among accessions revealed genetic divergence between the two species was much higher than that within species. Furthermore, we screened out 3 primers, in which ESGS79 and ESGS155 are able to amplify the specific bands to E.nutans accession, while Xgwm311 could amplify specific bands to E.sibiricus. These primers will provide guidance and convenience for the wild germplasm collection and breeding.