Bletilla striata (Thunb.) Rchb. f. is an endangered plant in China with important medicinal value. In this study, the genomic sequence library and microsatellite library of B. striata was set up based on Illumina sequencing. SSR primers were designed and carried out to test their polymorphism with 80 individuals from 4 wild populations. Four related species of B. striata were used to investigated their crosstaxa transferability. In addition, genetic diversity and genetic differentiation of the species were also investigated. The results showed that: (1) Microsatellite sequences were abundance in the genome of B. striata, 17 841 loci were detected in total.100 pair of primers were designed, 20 of them performed high polymorphism after PCR and polyacrylamide gelelectrophoresis, the alleles ranged from 2 to 6 with average of 3.85 per locus. The crosstaxa transferability was also examined in 4 related species, most of 20 loci were proved amplifiable in these species. (2) The study revealed that the genetic diversity of B. striata was high (N_a=3.85, I=1.07, H=0.614 7), genetic differentiation was prominent among populations (G_st=0.43). The gene flow among populations was limited (N_m=0.867 6), the adjacent populations performed tight genetic relationship as revealed by cluster analyses of populations. The reasons for genetic construction and conservational strategies of the species were also discussed in this study.