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Simple sequence repeats (SSRs) or microsatellites are an important class of molecular markers for genome analysis and plant breeding applications. In this paper, the SSR distributions within ESTs from the legumes soybean (Glycine max, representing 135.86 Mb), medicago (Medicago truncatula, 121.1 Mb) and lotus (Lotus japonicus, 45.4 Mb) have been studied relative to the distributions in cereals such as sorghum (Sorghum bicolor, 98.9 Mb), rice (Oryza sativa, 143.9 Mb) and maize (Zea mays, 183.7 Mb). The relative abundance, density, composition and putative annotations of di-, tri-, tetra- and penta-nucleotide repeats have been compared and SSR containing ESTs (SSR-ESTs) have been clustered to give a non-redundant set of EST-SSRs, available in a database. Further, a subset of such candidate EST-SSRs from sorghum have been tested for their ability to detect polymorphism between Striga-susceptible, stay-green drought tolerant mapping population parent 'E 36-1' and its Striga-resistant, non-stay-green counterpart 'N13'. Primer sets for 64% of the EST-SSRs tested produced a clear and specific PCR product band and 34% of these detected scorable polymorphism between the N13 and E 36-1 parental lines. Over half of these markers have been genotyped on 94 RILs from the (N13 x E 36-1)-based mapping population, with 42 markers mapping onto the ten sorghum linkage groups. This establishes the value of this database as a resource of molecular markers for practical applications in cereal and legume genetics and breeding. The primer pairs for non-redundant EST-SSRs have been designed and are freely available through the database (http://intranet.icrisat.org/gt1/ssr/ssrdatabase.html).