Twelve species, including three Hystrix species, five Leymus species, Hordeum bogdanii, Pseudoroegneria spicata, Psathyrostachys huashanica, and Roegneria ciliaris, were used for expressed sequence tag-polymerase chain reaction (EST-PCR) assay. A total of 125 products were amplified by 72 sets of EST-PCR markers developed in barley, among which 106 (84.8%) products were found to be polymorphic. Each EST-PCR marker produced 0-6 polymorphic bands, with an average of 1.47. The relationship between H. duthiei ssp. duthiei and H. duthiei ssp. longearistata is close, but they are remote to H. patula. Hystrix duthiei ssp. duthiei and H. duthiei ssp. longearistata were clustered with Leymus species and Psathyrostachys huashanica, which suggested that they have close genetic relationships. The results of EST-PCR analysis are basically comparable with those obtained from studies on cytology, which indicated that EST-PCR can be used to assess the genetic relationships among the perennial species in Triticeae.