Melilotus is one of the most important legume plants, in part due to its production of coumarin, but the genetic diversity among the 18 species remains unclear. In the present study, the analysis of genetic diversity of the Melilotus species was performed with simple sequence repeat (SSR) markers. From the PCR amplification, 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 SSR markers.