Expressed sequence tags (ESTs) are important resources for gene discovery and molecular marker development. From over 147,000 ESTs of Medicago truncatula, we have identified 4,384 ESTs containing perfect simple sequence repeats (EST-SSR) of di-, tri-, tetra- or pentanucleotides. Six hundred sixteen primer pairs (PPs) were designed and screened over a panel of eight genotypes representing six Medicago spp. and subspecies. Nearly, 74% (455) of the PPs produced characteristic SSR bands of expected size length in at least one Medicago species. Four hundred six (89%) of these 455 PPs produced SSR bands in all eight genotypes tested. Only 17 PPs were M. truncatula -specific. High levels of polymorphism (>70%) were detected for these markers in alfalfa, M. truncatula, and other annual medics. About 48% of the reported markers are part of gene transcripts linked to putative functions. Our results indicate that the SSR markers developed from M. truncatula ESTs are valuable genetic markers for the Medicago genus. These markers will be useful in establishing the genomic relationships of M. truncatula to important forage legume crops such as alfalfa and other annual medics.