EST-SSRs, a new kind of microsatellites (SSRs), was derived from expressed sequence tags (ESTs). A total of 597 EST-SSRs primer pairs were developed in this study, of which about 80%, 65.8%, 62.1% and 58.1% amplified products successfully in wheat, maize, rice and soybean, respectively. A total of 255 primer sets (42.7%) worked across the four species. Sequence similarity alignment of the 255 universal ESTs containing SSRs against database in GenBank indicated that 63 ESTs (30%) were associated with protein destination, and the rest were involved in metabolism, transcription, cellular development, defense system and other pathways related. It is of significance that these EST-SSRs markers will be highly valuable not only for wheat genomics also for comparative genomics among species.