8 Pags.- 2 Figs.- 1 Tabl. The definitive version is available at: https://www.actahort.org/index.htm Trees for commercial production comprise the fruit-bearing scions grafted to a rootstock, thus the choice of an appropriate rootstock is fundamental to orchard success. Even though several SNP-based linkage maps for cultivated Prunus species such as peach, cherries and almond are available, there are no SNP-based linkage maps for Prunus rootstocks. Here, we report on using a Genotyping-by-sequencing (GBS) approach to simultaneously discover and genotype SNPs from two peach rootstocks (‘Adafuel’ × ‘Flordaguard’) and their progeny. The plant material was developed at the EEAD-CSIC Prunus rootstocks breeding program with the goal to establish segregating progeny for a range of characters of importance to rootstock breeding (resistance to iron-chlorosis, root-asphyxia and replant diseases, as well as vigor and other effects on scions such as fruit quality and yield efficiency). High-density parental linkage maps were constructed by genotyping 118 siblings. Sequence reads obtained from GBS were aligned to the P. persica (L.) Batsch reference genome (Peach v2.0). A total of 18,912 high quality SNPs (MAF>0.05; missing data <5%), evenly distributed over the eight chromosomes, were identified. The number of identified SNPs ranged from 1,595 for the chromosome 7 to 4,042 for the chromosome 1. While eight linkage groups were constructed for ‘Adafuel’, only four linkage groups were constructed for ‘Flordaguard’, given the low heterozygosity of this genotype. High synteny and co-linearity was observed between obtained maps and Peach v2.0. These linkage maps of two phenotypically diverse Prunus rootstocks provide valuable information and will serve as the basis for identification of QTLs and genes relevant for Prunus rootstocks breeding. This work was funded by grants AGL2014-52063-R and INIA RFP 2015-00019, with FEDER and Gobierno de Aragón-A44 cofunding from Spain. It was also funded by CONICYTREGIONAL/GORE O´HIGGINS/CEAF/R08I1001, FONDECYT 3160316 and CONICYT R16F20006 from Chile. Carolina Font i Forcada is beneficiary of a Juan de la Cierva Incorporación 2017 contract. Peer reviewed