Drought is a major stress factor for agricultural production including alfalfa production. One way to counterbalance the yield losses is the introgression of drought tolerant germplasm into breeding programs. As an effort to exploit such germplasm, 16 individual plants were selected from the Southeastern Turkey from their natural habitat and clonally propagated in field trials with an ultimate goal to use the germplasm as parents for releasing a synthetic cultivar. Forage yield and forage quality traits were evaluated and molecular genetic diversity among genotypes were determined using inter simple sequence repeat markers. Genotypes showed a variation from growth habit to yield and quality traits indicating sufficient phenotypic variation for diverse breeding efforts (for grazing or harvesting) and long term selection schemes. A large amount of genetic variation was observed even with a limited number of marker and genotypes. However, no pattern of spatial genetic structure was observed for the scale of the study when genetic variation is linked to the geographic origin. We conclude that ex situ natural variation provides a wealth of germplasm that could be incorporated into breeding programs aiming to improve drought tolerance. We also suggest an extensive collection of seeds/plant tissue from unique plants with desirable traits rather than putting more efforts to create a spatial germplasm sampling efforts in narrow regions.