The main objective of this study is to produce DNA markers of polymorphism between wheat and some important alien species. The applicability of 1,165 barley EST primer sets to amplify markers showing polymorphism between wheat and ten alien species, covering a wide range of variation in Triticeae, was investigated. These primers consisted of four series. Series 1 was randomly chosen from a pool of barley EST primer sets. The remaining three were pre-screened in a previous study and showed polymorphic and co-amplified patterns between barley and wheat. From 22% to 100% of the primer sets amplified single clear bands in the species; from 29% to 75% of which were polymorphic to wheat. The frequency of amplification corresponded with their phylogenetic distance from barley. Many DNA markers (78–859) showing polymorphism between each species and wheat were obtained. These markers are expected to be valuable in identifying the alien chromosomes added to a wheat genetic background. The usefulness of the markers in the basic and the applied studies of the wild species is discussed.