Abstract: The aim of this study is to analyze the morphological traits of button medic genotypes and determine their molecular characterization using ISSR methods. Vegetative traits were assessed at 50% anthesis period, however, generative trait was evaluated at physiological period. Molecular diversity was determined by using 14 ISSR primers. To assess the genetic diversity and the genetic structure of wild button medic (Medicago orbicularis L.) collected from seventeen ecologically and geographically different locations of Southeastern Anatolian Region were analyzed by using ISSR at the Biotechnology Laboratory of Çukurova University. According to Jaccard’s similarity index, similarity between genotypes ranged from 0.63-1, with an average 0.70. According to dendrogram obtained from the similarity index data, the 17 genotypes clustered into two distinct groups, A and B. Group A was divided in to two (A1 and A2) sub-groups. Kulp, Diyarbakır, Eğil-1, Eğil-2, İdil and Ovabağ fell in to the sub-group of A1. In A2 group, one group was Silvan, Hilvan, Adıyaman, Derik, Malabadi and Erdurağı. Gaziantep, Cermik, Ergani and Palanlı samples categorized in to sub-group A2. The results obtained from this study will be helpful for button medic breeders in Turkey to gain information about genetic diversity and will enable them to make a future strategy for broadening the genetic basis of these crops.