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<p>Abstract</p> <p>Background</p> <p><it>Hordeum chilense</it>, a native South American diploid wild barley, is a potential source of useful genes for cereal breeding. The use of this wild species to increase genetic variation in cereals will be greatly facilitated by marker-assisted selection. Different economically feasible approaches have been undertaken for this wild species with limited direct agricultural use in a search for suitable and cost-effective markers. The availability of Expressed Sequence Tags (EST) derived microsatellites or simple sequence repeat (SSR) markers, commonly called as EST-SSRs, for barley (<it>Hordeum vulgare</it>) represents a promising source to increase the number of genetic markers available for the <it>H. chilense </it>genome.</p> <p>Results</p> <p>All of the 82 barley EST-derived SSR primer pairs tested for transferability to <it>H. chilense </it>amplified products of correct size from this species. Of these 82 barley EST-SSRs, 21 (26%) showed polymorphism among <it>H. chilense </it>lines. Identified polymorphic markers were used to test the transferability and polymorphism in other Poaceae family species with the aim of establishing <it>H. chilense </it>phylogenetic relationships. <it>Triticum aestivum</it>-<it>H. chilense </it>addition lines allowed us to determine the chromosomal localizations of EST-SSR markers and confirm conservation of the linkage group.</p> <p>Conclusion</p> <p>From the present study a set of 21 polymorphic EST-SSR markers have been identified to be useful for diversity analysis of <it>H. chilense</it>, related wild barleys like <it>H. murinum</it>, and for wheat marker-assisted introgression breeding. Across-genera transferability of the barley EST-SSR markers has allowed phylogenetic inference within the Triticeae complex.</p>