Fourteen microsatellite primer pairs were developed from a cDNA library of heat-treated seedlings of Fragaria vesca cv. yellow wonder. Transferability to 13 species of Fragaria ranged from 71% in diploid species F. gracilis Losinsk., F. iinumae Makino, F. nilgerrensis Schltdl. ex J. Gay and F. nipponica Makino, to 100% in octoploid domestic strawberry and its progenitors. Polymorphism was high in polyploid Fragaria species. However, polymorphism and heterozygosity of eight EST-SSRs (expressed sequence tag–simple sequence repeats) was low in 14 F. vesca genotypes.