The genetic structure of sweet chestnut (Castanea sativa Mill.) across Europe was assessed using 73 inter-simple sequence repeat markers to screen 1,768 individuals from 68 stands distributed across 29 sites in five European countries (Italy, France, Spain, Greece, and UK). At each site, trees were sampled from three distinct management types (domestication levels): naturalized stands, managed coppice, and grafted fruit orchards. In more than a third of the orchards, nonlocal genetic material (grafted clones) were evident, showing (as predicted) large differences from the other two domestication levels for most of the within-population genetic diversity parameters estimated. Randomly generated linkage disequilibrium analysis revealed weak though significant differences in two-locus allelic correlations between naturalized stands and coppice, suggesting that long-term management techniques may influence the genetic makeup of the populations. Multivariate analysis revealed the existence of five distinct gene pools across the study area; three were located in Greece, one on the northwestern coast of the Iberian peninsula and a large gene pool covering the rest of the Mediterranean basin. The implications of the results are discussed in relation to developing conservation strategies for chestnut genetic resources in Europe.