Tupistra chinensis Baker, belonging to Tupistra KerGawl, Liliaceae, is a perennial herb plant and a rare medicinal plant in China with sparse wildtype resource. In this study, the genome library and microsatellite library of T. chinensis were established for the first time based on the secondgeneration sequencing technology. Then its microsatellite composition was characterized. A set of simple sequence repeat (SSR) primers was developed, and T. chinensis and its close ribs were treated by PCR amplification and polyacrylamide gel electrophoresis detection. It was found that: (1) the genome sequencing and splicing returned 23 362 gene sequences and 1 465 microsatellite loci. In particular, the mononucleotides were the most popular, while the repetition lengths of dinucleotides were the most variable. (2) Fourteen out of 52 pairs of newlydesigned microsatellite primers were amplified into clear strips with rich polymorphism; the number of multiple alleles per locus (N_a) was 2-4 (mean 3.00). The polymorphism information content (PIC) was 0.336 9-0.785 6 (mean 0.546 3). (3) The 13 pairs of polymorphism primers asscreened were used to detect the generality of 6 close sibs of T. chinensis, including T. grandistigma, T. delavayi, T. wattii, T. ensifolia, Reineckia carnea, and Rohdea japonica. It was found the majority of the primers could be successfully amplified to the close ribs of T. chinensis, showing high generality. The microsatellite molecular markers of T. chinensis developed here underlie its genetic diversity research and germplasm resource identification in the future.