In order to strengthen the conservation and utilization of the germplasm resources of Fagus longipetiolata in Guizhou province, this study developed SSR molecular markers to analyze the genetic diversity and construct fingerprints of 100 germplasm resources collected from six natural populations. The results showed that the 12 pairs of polymorphic SSR primers were all chloroplast genome SSRs (cpSSRs), detecting a total of 136 alleles. The mean polymorphism information content (PIC) of the cpSSR loci was 0.767, showing high genetic polymorphism. The average observed allele (Na), average number of effective alleles (Ne), and average Shannon's index (I) of the six populations were 5.125, 3686, and 1.356, respectively. These indicated that the genetic diversity of the Fagus longipetiolata germplasm resources were relatively rich. Among them, the Duyun population had the highest genetic diversity (I = 1.589). Moreover, the ANOVA showed that the genetic variation was mainly from within the population (93%), which was consistent with the results of the coefficient of genetic differentiation between populations (Fst = 0.091). It was possible that the higher gene flow (Nm = 2.490) constrained the differentiation between populations. The 100 germplasm resources were divided into two subgroups based on population structure analysis, with the vast majority of them exhibiting high genetic purity (Q ≥ 0.6). The PI and Pisibs values were 1.11×10_-9 ^{and 3.74×10}_-6^{, respectively}. These indicated that the molecular marker fingerprinting developed in this study had a strong discriminatory ability. The DNA fingerprints of the 100 germplasm resources were constructed based on five pairs of core primers. This study provided scientific basis and technical support for the identification, preservation, breeding and parentage tracing of Fagus longipetiolata in Guizhou province.