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This study presents the results of the first genetic analysis of ancient chestnut trees (Castanea sativa Mill.) in Italy and in the Iberian Peninsula to better understand the effect of grafting on the domestication process of chestnut and to investigate the impacts of early selection and improvement on the genetic diversity retained. We evaluated 105 giant ancient trees from Italy, Spain and Portugal and compared them with the European Union (EU) database of chestnut cultivars by using a set of 24 simple sequence repeats (SSRs; microsatellite markers). We measured the perimeter (girth) at the diameter at breast height (DBH). Samples from both the canopy and the roots of each tree were analysed to distinguish which trees were self-rooted and which were grafted. Diversity was compared using standard metrics and model-based approaches based on the expected heterozygosity (He) at equilibrium. We could differentiate 91 new genotypes; 9.6% matched known chestnut cultivars. We found the first evidences of cultivation, that is, grafting to produce “instant domestication” in Galicia and in the Douro Valley in trees of 14-m perimeter (15th century) and in the Basque Country (first report in that area) in a tree of 11.5-m perimeter (16th century). In Italy, the cultivar “Marrone Fiorentino” was found in some giant trees with perimeters of 8 and 9 m (17th-18th centuries) in the Toscana and Umbria. Those findings matched with written references in Portugal from the 16th century and from the 18th century in Spain. “Instant domestication” could be dated back to the 15th century and was related to the wild populations existing in the same areas where cultivars are being propagated, without a different genetic structure for wild chestnut trees and with a high diversity maintained through the initiation of domestication.