Technical Abstract: Orchardgrass or cocksfoot [Dactlyis glomerata (L.)] has been naturalized on nearly every continent and is a commonly used species for forage and hay production. All major cultivated varieties of orchardgrass are auto-tetraploid, and few tools or information are available for functional and comparative genetic analyses and improvement of the species. To improve the genetic resources for orchardgrass, we have developed an EST library and SSR markers from salt, drought, and cold stressed tissues. The ESTs were bi-directionally sequenced from clones and combined into 17,373 unigenes. Unigenes were annotated based on putative orthology to genes from rice, Triticeae grasses, other Poaceae, Arabidopsis, and the non-redundant database of the NCBI. Of 1,162 SSR markers developed, approximately 80% showed amplification products across a set of orchardgrass germplasm, and 40% across related Festuca and Lolium species. Polymorphic information content on a subset of 33 SSR markers ranged from 0.18 to 0.42, and when orchardgrass subspecies were genotyped using these SSR markers, their within-accession similarity values ranged from 0.44 to 0.71. The total number of genotyped bands was greater for tetraploid accessions compared to diploid accessions, and accessions tended to group by ploidy level before subspecies designation. Clustering analysis indicated grouping of Mediterranean subspecies and central Asian subspecies, while the D. glomerata ssp. aschersoniana was closest related to three cultivated varieties.