[Objective] The genetic variations of ancient chestnut trees in Ming and Qing dynasties were studied based on molecular markers. [Methods] 33 accessions were collected from Bohai town at Huairou district of Beijing, and the genetic diversity and polymorphism were detected by using 13 fluorescent polymorphic simple sequence repeats (SSRs). [Results] Thirteen SSR primers amplified 61 polymorphic alleles and 81 amplified bands in the 33 ancient chestnut trees, the number of alleles per locus ranged from 2 to 11 with an average of 4.7, and the amplified bands of 13 pairs of primers were 2 to 16 with an average of 6.2. The mean expected heterozygosity (HE) and polymorphic information content (PIC) were 0.4528 and 0.4103, respectively. PRA 75, PRA 83, PRD 25, CsCAT7 and PRD 53 can distinguish all of the ancient chestnut trees. [Conclusion] The habitats of chestnut trees in Ming and Qing dynasties were identical, the gene flow and gene exchange existed in a small area, which may be closely related to the low genetic diversity level. The study provides references for genetic diversity protection and utilization of chestnut germplasm resource. And the amplified bands and peak figures of 33 chestnut trees could lay the foundation for germplasm identification of the chestnut trees in Ming and Qing dynasties.