Abstract: During the investigation and collection of Bletilla striata resources, it was found that many characteristics of Bletilla striata such as flower type, flower color, florescence, leaf shape and so on had great variation degrees. In this study, we collected 30 accessions of Bletilla striata resources from Sichuan and Yunnan province, cloned and analyzed their nrDNA ITS and chloroplast ycf1 gene differences, and found that ycf1 gene has little difference and ITS gene has large difference. According to the differences of ITS sequences, Bletilla striata was clustered into 3 branches. Combined with the apparent characteristics of Bletilla striata, it was found that the ITS sequences of Bletilla striata had obvious correlation with florescence and leaf type, but had no obvious correlation with other characteristics such as flower type and color. This study provided a theoretical basis for the collection, development and utilization of Bletilla striata germplasm resources.