Our previous results showed that the interspecific relation-ships within the Melilotus genus based on the phylogenetic tree are clearly monophyletic in the legume family (Di et al., 2015).A preliminary evaluation of agronomy and quality traits of 19 Melilotus accessions showed that coumarin content could vary from 0.16–1.02% (Luo et al., 2014). In this study, the SSR markers were used to study the genetic diversity and population structure among50 accessions of 18 Melilotus species, to talling 326 plant samples that were collected. The SSR markers founded in this study will be a useful resource for genetic study and germplasm evaluation for coumarin production in the Melilotus genus.