DNA markers have been studied and used intensively to identify plant species based on molecular approaches. The genus Medicago belongs to the family Fabaceae and contains 87 species distributed from the Mediterranean to central Asia. Five species of Medicago are known to be distributed in South Korea; however, their morphological characteristics alone cannot distinguish the species. In this study, we analyzed the phylogenetic relationships using collected five species of Medicago from South Korea and 44 taxa nucleotide information from NCBI. The constructed phylogenetic tree using gibberellin 3-oxidase 1 and tRNALys (UUU) to maturase K gene sequences showed the monophyly of the genus Medicago, with five species each forming a single clade. These results suggest that there are five species of Medicago distributed in South Korea. In addition, we designed polymerase chain reaction primers for species-specific detection of Medicago by comparing the plastid sequences. The accuracy of the designed primer pairs was confirmed for each Medicago species. The findings of this study provide efficient and novel species identification methods for Medicago, which will assist in the identification of wild plants for the management of alien species and living modified organisms.