The increasing availability of expressed sequence tags (ESTs) in wheat (Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. In this study, 300 primer pairs were designed from 265 wheat ESTs that contain microsatellites in order to develop new markers for wheat. Their level of transferability in eight related species [Triticum durum, T. monococcum, Aegilops speltoides, Ae. tauschii, rye (Secale cereale), barley (Hordeum vulgare), Agropyron elongatum and rice (Oryza sativa)] was assessed. In total, 240 primer pairs (80%) gave an amplification product on wheat, and 177 were assigned to wheat chromosomes using aneuploid lines. Transferability to closely related Triticeae species ranged from 76.7% for Ae. tauschii to 90.4% for T. durum and was lower for more distant relatives such as barley (50.4%) or rice (28.3%). No clear putative function could be assigned to the genes from which the simple sequence repeats (SSRs) were developed, even though most of them were located inside ORFs. BLAST analysis of the EST sequences against the 12 rice pseudo-molecules showed that the EST-SSRs are mainly located in the telomeric regions and that the wheat ESTs have the highest similarity to genes on rice chromosomes 2, 3 and 5. Interestingly, most of the SSRs giving an amplification product on barley or rice had a repeated motif similar to the one found in wheat, suggesting a common ancestral origin. Our results indicate that wheat EST-SSRs show a high level of transferability across distantly related species, thereby providing additional markers for comparative mapping and for following gene introgressions from wild species and carrying out evolutionary studies.