Stone fruit (Prunus L.) production in Chile covers approximate to 43,000 ha and includes a wide variety of soils and climates requiring a large diversity of rootstocks. The most commercially important rootstock cultivars are 26 genotypes from three different taxonomic groups belonging to the subgenera Amygdalus (L.) Benth. Hook. (peach group), Prunus Focke [= Prunophora (Neck.)] Focke (plum group), and Cerasus (Adans.) Focke (cherry group) with eight, seven, and 10 individuals, respectively. To determine their genetic diversity, characterization by microsatellite markers [simple sequence repeat (SSR)] was conducted. Of a total of 20 SSR markers evaluated, 12 generated amplified products that were consistent in the three taxonomic groups. The number of alleles per marker ranged from 18 for PSM-3 to four in CPPCT-002. Clustering analysis, by both traditional hierarchical and model-based approaches, indicate that all genotypes are clustered in their respective taxonomic groups, including the interspecific hybrids. Genetic diversity, measured as the average distances (expected heterozygosity) between individuals in the same cluster, was higher in Cerasus (0.78) followed by Prunus (0.72) and Amygdalus (0.64). Total number of alleles observed was 133, of which 14, 33, and 35 from six, 10, and 10 loci were unique for the peach, plum, and cherry rootstock groups, respectively. Alleles shared among peach/plum, plum/cherry, and peach/cherry rootstock genotypes were 13, 14, and 18 from nine, seven, and seven loci, respectively. Only six alleles from five loci were common to the three taxonomic groups. In addition, to develop a rootstock identification system based on SSR markers, a minimum set of three markers (PMS-3, BPPCT-037, and BPPCT-036) able to differentiate the 26 genotypes was identified. This study is the first step toward establishing a stone fruit rootstock breeding program in Chile.