Modern plant breeding utilizes a large number of molecular markers for segregation analysis, gene mapping and marker discovery to facilitate marker-assisted selection (MAS). Genotyping-by-sequencing (GBS) is a cost-effective next-generation sequencing technology that provides a platform for detection of a large number of single nucleotide polymorphisms (SNPs) in a large sample pool. The Centro de Estudios Avanzados en Fruticultura (CEAF), located in Rengo, Region de O'Higgins, Chile, initiated a Prunus rootstock breeding program in 2010. Consequently, different interspecific crosses have been carried out utilizing the Prunus rootstock germplasm collection established at CEAF and available pollen obtained from Prunus rootstock germplasm of the Spanish Estacion Experimental de Aula Dei-Consejo Superior de Investigaciones Cientificas (EEAD-CSIC). In the present study, 53 diploid Prunus rootstocks and five scion cultivars were genotyped using GBS for genome-wide SNP identification, enabling the assessment of the genetic diversity of both Chilean and Spanish germplasm collections. A group of 45,391 high-quality SNPs (minor allele frequency (MAF) > 0.05; missing data < 5%) were selected for further analysis of this group of 58 genotypes. These SNPs are distributed in genic and intergenic regions of each chromosome, with an average of 50% located in exonic regions. The genetic diversity detected among the studied accessions divided them into three groups that are in agreement with their current taxonomic classification. These results demonstrate the strong potential of SNPs identified from GBS for Prunus rootstock identification and to study genetic diversity in Prunus species. Also, given the large number of SNPs identified in exonic regions, this strategy represents an important tool for finding candidate genes that underlie traits of interest and potential functional markers for use in MAS.