The Rosodae supertribe is composed of economically important species (strawberry, rose and raspberry). In order to extend the repertoire of markers for optimizing genetic mapping and synteny within this supertribe, we developed 20 functional markers from two Fragaria cDNA libraries enriched in AG/AC microsatellite motifs. We tested their transferability within the Rosodae supertribe, including two reference diploid and octoploid strawberry segregating populations. Out of these 20 functional markers, 70% revealed polymorphism in both diploid and octoploid Fragaria segregating populations and were mapped on the diploid genetic map to provide useful information for further marker-assisted selection. These 20 EST-SSRs were also tested in 17 wild Fragaria species and displayed a high cross-species transferability (mean of 95%), providing new markers for further diversity studies within the genus. These markers were also transferable to the phylogenetically close Potentilla genus, whereas their transferability was lower for the polyploid Duchesnea indica species and for the more distant Rosa genus. Their use for genetic mapping within the Rosodae will further permit a comparison of genetic information among the different genera.