Licorice (Glycyrrhiza glabra) is an important medicinal herb and has long been used in traditional medicine for the treatment of several diseases worldwide. Understanding the genetic diversity within Glycyrrhiza species is important for the efficient conservation of these medicinal herbs. In this study, we have developed 20 polymorphic chloroplast microsatellite (cpSSR) markers using the chloroplast genome of G. lepidota. The cpSSR markers were tested on a total of 27 Glycyrrhiza individual plants. The number of alleles per locus ranged from two to eight among the Glycyrrhiza accessions. Overall, the Shannon index (I) for each cpSSR ranged from 0.315 to 1.694, the diversity indices (h) were 0.140–0.793 and the unbiased diversity indices (uh) were 0.145–0.825. In addition, the cpSSR markers were successfully divided and classified the 27 Glycyrrhiza individuals into four groups. The cpSSR markers developed in this study could be used in the assessment of genetic diversity and rapid identification of Glycyrrhiza species.