This work investigated the genetic diversity of 134 Castanea sativa Mill. accessions present in the Italian region of Emilia-Romagna. Samples were taken from three collection fields (Granaglione, Zocca and Paloneta) in the Tuscan-Emilian Apennines. The accessions were analyzed by using 16 microsatellite markers (SSR). Genetic distances among accessions, calculated through the DICE coefficient, were used to construct an UPGMA cluster analysis. One major genotype (named “Marroni”) was identified across the three investigated collection fields; this variety corresponds to a sweet chestnut cultivar that has been propagated and widely diffused in the Emilia-Romagna region. Other genotypes were represented by different varieties of Italian chestnuts. The results of this study will be used to define and share guidelines for the characterization and varietal certification of the chestnut varieties in the Emilia-Romagna region.