In this study, 14 phenotypic traits including single grain weight, cross-diameter length and longitudinal diameter length of 55 chestnut cultivars were measured. However, no significant differences on morphological traits in subsets of chestnut varieties were observed, and it is difficult to identify the varieties accurately using phenotypic traits. We designed SSR markers based on chestnut genome, followed by PCR amplification of 55 chestnut species and data analysis using the software POPGENE32, PowerMarker V3.25 and NTSYS V2.10. By taking use of six pairs of SSR primers with good polymorphisms, 31 alleles were detected and the percentage of polymorphic loci was 100%. The mean values of polymorphism information content index, Nei’s gene diversity index, Shannon’s polymorphism information index, observed heterozygosity and expected heterozygosity were 0.651, 0.696, 1.358, 0.524 and 0.703, respectively. According to the SSR-based genotyping results, 55 chestnut cultivars were classified enabling the construction of their fingerprints. Collectively, this study deployed the phenotypic datasets and SSR markers to decipher the genetic diversity of chestnut varieties and generate the molecular ID cards, which provided theoretical references for the variety identification, rational utilization and effective protection of chestnut germplasm resources.