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Agropyron cristatum  is a wild grass of the tribe Triticeae and serves as a gene donor for wheat improvement. However, very few markers can be used to monitor  A. cristatum  chromatin introgressions in wheat. Here, we reported a resource of large-scale molecular markers for tracking alien introgressions in wheat based on transcriptome sequences. By aligning  A. cristatum  unigenes with the Chinese Spring reference genome sequences, we designed 9602  A. cristatum  expressed sequence tag-sequence-tagged site (EST-STS) markers for PCR amplification and experimental screening. As a result, 6063 polymorphic EST-STS markers were specific for the  A. cristatum  P genome in the single-receipt wheat background. A total of 4956 randomly selected polymorphic EST-STS markers were further tested in eight wheat variety backgrounds, and 3070 markers displaying stable and polymorphic amplification were validated. These markers covered more than 98% of the  A. cristatum  genome, and the marker distribution density was approximately 1.28 cM. An application case of all EST-STS markers was validated on the  A. cristatum  6 P chromosome. These markers were successfully applied in the tracking of alien  A. cristatum  chromatin. Altogether, this study provided a universal method of large-scale molecular marker development to monitor wild relative chromatin in wheat.

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A resource of large-scale molecular markers for monitoring Agropyron cristatum chromatin introgression in wheat background based on transcriptome sequences