A size-selected genomic library from Elymus alaskanus was screened for the presence of (GA)n, (GT)n, (CAC)n, and (TCT)n microsatellite sequences. A total of 28 positive clones were found for the two dinucleotide repeats, whereas no positive clones were found for the trinucleotide repeats. Positive clones were sequenced to validate the presence of microsatellites and to generate polymerase chain reaction (PCR) primers, based on the sequences flanking the microsatellite. Primer pairs were designed and evaluated for 18 selected microsatellites. The resulting loci were analysed by PCR for their usefulness as molecular markers in an array of 18 accessions representing E. alaskanus and 10 other Elymus species. PCR amplification revealed alleles for the 18 loci, which varied in having 1-10 alleles in these accessions. In the 18 accessions tested, 7 of the 18 loci were polymorphic, with gene diversity values ranging from 0.54 to 0.80 among all species. Within E. alaskanus, gene diversity values ranged from 0.20 to 0.72, with a mean of 0.48. Polymorphism was also detected within accessions. No clear relationship between total repeat length and the degree of polymorphism was observed in this study. Primer pairs designed to amplify microsatellites in E. alaskanus can be used to generate polymorphism products in other species within the genus. Hence, microsatellites are useful markers for studying both inter- and intra-specific genetic variability within Elymus.