•We characterize by nSSR markers ancient chestnut widespread in Northern Italy.•The use of only three markers allowed to identify 28 distinct genetic profiles.•The results demonstrated a vast genetic variability within the population.•This variability will represent the starting point for a cultivar agronomical evaluation. The presence of the chestnut (Castanea sativa Mill.) in all Italian regions, within altitude limits depending on latitude, and in very different exposure and soil conditions, favored the formation of a rich patrimony of cultivars. In the course of several centuries of cultivation a number of genotypes appeared and were selected, also by virtue of a widespread habit of propagating the tree by seed; these genotypes differ for numerous traits, particularly for those relating to the fruit, but plant resistance to biotic and abiotic factors has been important in the selection process. However, the large existing genetic patrimony is increasingly at risk of genetic erosion and it is extremely important to study and valorize local germplasm in order to preserve the existing biodiversity and to identify potentially useful characters for a revival of the Italian chestnut industry. The purpose of this study was to characterize genetically, with eight nSSR (di- and trinucleotide, CsCAT and EMCs series respectively) markers, 54 ancient chestnut accessions widespread in a small area of Northern Italy. The set of chosen markers resulted as a whole highly polymorphic, and made possible the characterization of all studied accessions. The use of only three markers allowed the identification of 28 distinct genetic profiles. Data were analyzed by cluster analysis, and results demonstrated a vast genetic variability within the population. The nSSR used, moreover, allowed us to identify most unknown accessions and possible homonyms and synonyms, and cases of intravarietal clones within the local germplasm.