Microsatellites or simple sequence repeats (SSRs) are highly useful molecular markers for plant improvement. Expressed sequence tag (EST)-SSR markers have a higher rate of transferability across species than genomic SSR markers and are thus well suited for application in cross-species phylogenetic studies. Our objectives were to examine the amplification of tall fescue EST-SSR markers in 12 grass species representing 8 genera of 4 tribes from 2 subfamilies of Poaceae and the applicability of these markers for phylogenetic analysis of grass species. About 43% of the 145 EST-SSR primer pairs produced PCR bands in all 12 grass species and had high levels of polymorphism in all forage grasses studied. Thus, these markers will be useful in a variety of forage grass species, including the ones tested in this study. SSR marker data were useful in grouping genotypes within each species. Lolium temulentum, a potential model species for cool-season forage grasses, showed a close relation with the major Festuca–Lolium species in the study. Tall wheatgrass was found to be closely related to hexaploid wheat, thereby confirming the known taxonomic relations between these species. While clustering of closely related species was found, the effectiveness of such data in evaluating distantly related species needs further investigations. The phylogenetic trees based on DNA sequences of selected SSR bands were in agreement with the phylogenetic relations based on length polymorphism of SSRs markers. Tall fescue EST-SSR markers depicted phylogenetic relations among a wide range of cool-season forage grass species and thus are an important resource for researchers working with such grass species.Key words: phylogeny, EST-SSR, forage grasses, tall fescue.