Leymus chinensis, a major component of the plant community in the eastern Eurasian grasslands with a wide distribution, provides stability to grassland ecosystems and supports animal husbandry. This study aimed to bridge the gap between the molecular breeding and industrial application of L. chinensis by conducting a comprehensive simple sequence repeat (SSR) analysis. A total of 973,129 SSRs were identified in the L. chinensis whole genome, which was used to design 20 polymorphic pairs of SSR primers to further assess 105 L. chinensis accessions. On average, 33.55 alleles were detected per locus, with an average Shannon index of 2.939 and a polymorphic information content value of 0.910. Principal coordinate, maximum likelihood, and structure analyses consistently showed that all samples were coincidentally divided into four subclasses. In addition, Mantel test data indicated a weak correlation between genetic and geographical distances in L. chinensis, whose variability may be related to the pollination mode and natural selection pressures. Finally, we used the 20 pairs of selected markers to scan 105 accessions, constructing a fingerprint for them. These findings provide new foundations for identifying superior varieties, improving the management of genetic resources, and constructing a germplasm resource database for L. chinensis.