Abstract: Background and Objective: Koelreuteria as important native tree species, have multiple ornamental, ecological and medicinal values. However, research on the germplasm resources of Koelreuteria is relatively limited and relies primarily on the use of morphological differences to distinguish trees; thus the aim of this study was to develop effective EST-SSR markers to provide reference for the genetic diversity evaluation of the species. Methodology: In this study, 2871 microsatellite markers were assessed by mining a transcriptomic database of Koelreuteria paniculata Laxm. and 65,360 pairs of primers were designed. Based on the length of the products, the stability and permeability of primers and primer annealing temperatures, 96 pairs were screened and tested in a preliminary test. Thirteen pairs were selected for capillary electrophoresis analysis. The observed heterozygosity and expected heterozygosity were collected by GenAlEx software and the allelic polymorphism information content were calculated using CERVUS. Results: Ninety two pairs that gave clear bands in PCR amplification. For capillary electrophoresis analysis, in the microsatellite loci that showed polymorphisms, the number of alleles ranged from 2-5 per locus and the polymorphic information content ranged from 0.124-0.726, respectively. Additionally, based on capillary electrophoresis results, this study established unique fingerprints for four particular variants of K. paniculata Laxm. Conclusion: These polymorphic markers will be in a great favour of evaluating genetic biodiversity. The fingerprints would provide a foundation for variety protection.