Genotyping-by-Sequencing (GBS) was applied in a set of 53 diploid Prunus rootstocks and five scion cultivars from three subgenera (Amygdalus, Prunus and Cerasus) for genome-wide SNP identification and to assess genetic diversity of both Chilean and Spanish germplasm collections. A group of 45,382 high quality SNPs (MAF >0.05; missing data <5%) were selected for analysis of this group of 58 accessions. These SNPs were distributed in genic and intergenic regions in the eight pseudomolecules of the peach genome (Peach v2.0), with an average of 53% located in exonic regions. The genetic diversity detected among the studied accessions divided them in three groups, which are in agreement with their current taxonomic classification. SNPs were classified based on their putative effect on annotated genes and KOG analysis was carried out to provide a deeper understanding of the function of 119 genes affected by high-impact SNPs. Results demonstrate the high utility for Prunus rootstocks identification and studies of diversity in Prunus species. Also, given the high number of SNPs identified in exonic regions, this strategy represents an important tool for finding candidate genes underlying traits of interest and potential functional markers for use in marker-assisted selection.