Abstract: To estimate the allelic variation at the expressed sequence tag simple sequence repeats (EST-SSR) among Persian wheat (Triticum turgidum ssp. carthlicum L.) accessions collected from fifteen countries and provide information for wheat breeding and improvement in southwest China, 87 Persian wheat accessions from fifteen countries were investigated by using EST-SSR markers. EST-SSRs were molecular markers belonging to the transcribed region of the genome. Therefore, any polymorphism detected by EST-SSRs might reflect better relationship among species or varieties. Fourteen primer pairs could successfully amplify the fragments in the 87 accessions, of which tri-nucleotide repeats were the dominant type. A total of 33 eSSR alleles were detected, and the number of alleles detected by single pair primers ranged from 3 to 7 per locus, with an average of 3.71. Clustering analysis suggested that most of the accessions with adjacent geographic origins had the tendency to cluster together. Therefore, when used in Persian wheat genetic analysis, EST-SSR markers not only act as genetic markers but also reveal differences in related gene expression.