The microsatellite markers in Firminana danxiaensis were developed by taking use of from the transcriptome dataset. These markers were subjected for analyzing the genetic diversity of extant populations to provide basis for rational utilization and the protection of Firminana danxiaensis. Primer 3.0, GenAlEx6.3, FSTAT and MS-tools were deployed to explore primers and analyze the genetic parameters. As a result, 17,858 SSRs were identified from 79,920 unigenes with a frequency of 1/4.78kb. Trinucleotide, tetranucleotide and dinucleotide accounted for 43.64%, 23.52% and 15.54%, respectively. AAG/CTT and AG/CT were the most abundant repeat motif for trinucleotide and dinucleotide, respectively. Out of seventy-three primers that were tested in 16 genotypes, 16 primers were polymorphic with an average value of polymorphic information content (PIC) 0.546. The principal component analysis (PCA) showed that these loci are efficient to identify individuals from different regions and each individuals. A moderate genetic diversity among three Firminana danxiaensis populations and the expected heterozygosity (He) varied from 0.550 to 0.605 were observed. The geology, historical climate change and human disturbance may be the main reasons for Firminana danxiaensis deviated from Hardy-Weinberg equilibrium. Taken together, these newly developed EST-SSR markers will lay foundation for unlocking the genetic structure and the marker-assisted breeding of Firminana danxiaensis.