| Summary: | Background: <i>Elymus</i> L. is the largest genus in the Triticeae tribe. Most species in this genus are highly stress resistant, with excellent forage value. <i>Elymus breviaristatus,</i> a rare species endemic to the Qinghai-Tibet Plateau (QTP), is declining due to habitat fragmentation. However, genetic data for <i>E. breviaristatus</i> are limited, with expressed sequence tag (EST) markers being particularly rare, hampering genetic studies and protection measures. Results: We obtained 9.06 Gb clean sequences from the transcriptome of <i>E. breviaristatus</i>, generating 171,522 unigenes, which were assembled and functionally annotated against five public databases. We identified 30,668 SSRs in the <i>E. breviaristatus</i> transcriptome, from which 103 EST-SSR primer pairs were randomly selected. Of these, 58 pairs of amplified products of the expected size, and 18 of the amplified products were polymorphic. Model-based Bayesian clustering, the unweighted pair group method with arithmetic average (UPGMA), and principal coordinate analysis (PCoA) of 179 wild <i>E. breviaristatus</i> in 12 populations using these EST-SSRs were generally consistent, grouping the 12 populations into two major clades. Analysis of molecular variance (AMOVA) found 70% of the genetic variation among the 12 populations and 30% within the populations, indicating a high level of genetic differentiation (or low gene exchange) among the 12 populations. The transferability of the 58 successful EST-SSR primers to 22 related hexaploid species was 86.2–98.3%. UPGMA analysis generally grouped species with similar genome types together. Conclusions: Here, we developed EST-SSR markers from the transcriptome of <i>E. breviaristatus.</i> The transferability of these markers was evaluated, and the genetic structure and diversity of <i>E. breviaristatus</i> were explored. Our results provide a basis for the conservation and management of this endangered species, and the obtained molecular markers represent valuable resources for the exploration of genetic relationships among species in the <i>Elymus</i> genus.
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