EST-SSR from Medicago truncatula Gaertn. and Glycine max (L.) Merr. were tested for transferability in various species of Onobrychis (O. pyrenaica Sennen, O. argentea Boiss. and O. viciifolia Scop.). Repeatable amplification was obtained for 81% of the microsatellites and 52% were polymorphic. Six selected SSRs from M. truncatula were used to fingerprint and estimate the genetic similarity of a set of 23 accessions of O. viciifolia. PCA analysis discriminated among the different Onobrychis species and the sainfoin accessions were clustered in a single major group. This grouping is discussed in terms of the history of cultivation of sainfoin in Spain. The selected SSRs will allow fingerprinting and genetic studies in Onobrychis species, solving the lack of available SSR markers in this species.