In Switzerland, chestnut forests cover about 27,100 ha, plus some 6800 ha of mixed stands. Due to environmental and historical reasons, most of these still existing forests are located in the Swiss Southern Alps, whereas in the northern parts of the country the chestnut cultivation and the related knowledge strongly regressed since the Little Ice Age period. Nevertheless, Switzerland still hosts valuable genetic resources of the sweet chestnut tree. The present genetic study bases on a nationwide inventory, identification and precise localisation of old and/or grafted chestnut trees for conservation purposes. The main objectives were: (1) to evaluate the genetic diversity and the genetic structure of Castanea sativa in Switzerland, and (2) to define a program of conservation including the proposal of a defined core collection. We genetically analysed a pre-selection of 962 accessions (out of 14,165 inventoried trees throughout Switzerland), profiling them with 24 microsatellites. We identified 675 different genotypes out of 962 accessions with a 29.8% of repetitiveness due to clonality. A structural analysis based on a Bayesian method allowed to identify two main clusters, one mostly related to the genetic group from southern Europe (Reconstructed Panmictic Population RPP1) and a second one (RPP2) which revealed to be independent and genetically different from other European groups of chestnut cultivars. The Swiss RPP2 represents a new genetic group, and consequently a complement to genetic resources of chestnut tree in Europe. Genetic analysis allowed defining a core collection of 46 genotypes, which should be used in priority for the Swiss conservation program.