The morphological similarity of Elymus trachycaulus to the Eurasian E. caninus has often been noted. This has lead to controversial and contradicting taxonomic treatments. Nevertheless, there has been no systematic investigation on molecular genetic similarity between E. trachycaulus and E. caninus. In this study, random amplified polymorphic DNA (RAPD) analysis was used to study the similarity between the two species. RAPD analysis of 38 samples representing E. caninus and E. trachycaulus complex yielded 111 interpretable RAPD bands. The Jaccard's similarity values for E. caninus ranged from 0.38 between accessions H10345 and H10353 to 0.97 between accessions H8745 and H10096, with an average of 0.67. The Jaccard's similarity values for E. trachycaulus complex ranged from 0.09 between E. trachycaulus ssp. subsecundus (PI 537321) and E. trachycaulus ssp. violaceus (PI 272612) to 0.78 between accessions PI 315368 and PI 372644, with an average of 0.43. The results from different analyses (NJ and PCA) were similar but not identical. The molecular genetic separation between E. caninus and E. trachycaulus was consistent. The PCA analysis clearly separated all E. caninus accessions from E. trachycaulus and its subspecies. The NJ analysis also showed separation between most accessions of E. caninus and E. trachycaulus. Further analysis excluding E. trachycaulus ssp. subsecundus and ssp. violaceus revealed that E. caninus species and E. trachycaulus species were clearly separated into two distinct groups. The RAPD data thus support the treatment of E. caninus and E. trachycaulus as distinct species. The analyses further indicate that E. violaceus is nested within E. trachycaulus, and more related to E. trachycaulus complex rather than to E. caninus.