Abstract: The inter-simple sequence repeat (ISSR) method was used to study the genetic diversity and genetic structure of 138 switchgrass accessions to provide information on how to improve the utilization of switchgrass germplasms. 1) Sixteen primers were selected from 100 ISSR primers, and a total of 220 bands were amplified. Of these bands, 196 were polymorphic (89.09%) and the average number was 13.75 per primer. 2) POPGENE analysis showed that the average information index of Shannon (I) was 0.3880 and gene diversity of Nei (H) was 0.2498, indicating a rich genetic diversity of switchgrass germplasm resources. 3) Analysis of AMOVA showed genetic variance within an ecotype was larger than that between ecotypes, while the genetic variance within an ecotype and between ecotypes was 56.60% and 43.40%, respectively. 4) Using NTsys-pc V2.1 software, the 138 accessions were clustered into two groups (lowland and upland ecotypes) by UPGMA clustering and principal component analysis (PCA) with the similarity coefficient ranging from 0.4000 to 0.8818 (average of 0.7237). In the upland ecotype, six subgroups were distinguished. Some cultivars and populations could not be grouped according to their pedigrees or collection area.