Recent increase in the availability of expressed sequence tag (EST) data has facilitated the development of microsatellite or simple sequence repeat (SSR) markers in a number of plant species groups, including cereals. As these SSRs are derived from ESTs/genes (EST-SSRs), they exhibit a higher potential for transfer through cross-amplification in related species than SSR markers generated from genomic DNA libraries. In this study, a sub-set of 165 EST-SSR markers from a total of 185 assigned to the genetic map of barley was examined for transferability to wheat, rye and rice. A higher proportion, i.e., 78.2% of barley markers showed amplification in wheat followed by 75.2% in rye and 42.4% in rice. Furthermore, in silico comparison of SSR-ESTs (ESTs containing SSRs) corresponding to 185 mapped barley EST-SSR loci against 1,369,182 publicly available cereal ESTs showed significant homology with ESTs of wheat (93.5%), rye (37.3%), rice (57.3%), sorghum (51.9%) and maize (51.9%). Sequence similarity of the barley ESTs with 379,944 ESTs of the two model dicot species, Arabidopsis and Medicago suggested theoretical transferability of barley markers into dicot species although at low frequency (9.7% in Arabidopsis and 8.6% in Medicago). In silico comparative mapping (sequence comparison) of mapped barley SSR-ESTs against the mapping data of rye, wheat and rice indicated the presence of orthologues of the barley SSR-ESTs in the respective species. Furthermore, nine barley EST-SSRs were experimentally mapped to a rye genetic linkage map and all could be located in the expected orthologous region compared to their position in barley.