tMelilotus is one of the most important legume plants, in part due to its production of coumarin, butthe genetic diversity among the 18 species remains unclear. In the present study, the analysis of geneticdiversity of the Melilotus species was performed with simple sequence repeat (SSR) markers. From the PCRamplification, we selected 18 out of 70 primers that were used in previous studies. Three hundred twenty-six sampled individuals were assayed to study the genetic diversity and polymorphisms based on the SSRmarkers. All analyzed markers were polymorphic, and 287 alleles were identified, with 15.94 alleles perlocus detected. The polymorphism information content (PIC) values ranged from 0.71 to 0.93, with anaverage of 0.87, which indicates that the markers were highly informative. Based on the unweighted pair-group method with arithmetic mean (UPGMA) cluster analysis, we found that the 18 species were dividedinto two clusters. M. italicus and M. speciosus from cluster A and M. indicus and M. segetalis from clusterB were closely related. Population structure analysis suggested that the optimum number of groups wasthree. From the analysis of molecular variance, 17.79% of the variance was due to species differences,31.61% of the variance was due to differences among populations within species and the remaining50.60% was due to differences within populations. The results of the present study showed that theseSSR markers will benefit the Melilotus research community for genetics and breeding. Furthermore, thisstudy also established the foundation for future breeding programs, genetic improvement and coumarinproduction in the Melilotus species.