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Medicago ruthenica (L.) Ledebour, a species well adapted to dry, stony and desert habitats, has more tolerance to drought, cold and saline conditions than other alfalfas. Characterization of the genetic diversity and population genetic structure of M. ruthenica is important for its domestication as a legume forage species and for the development of other cultivated alfalfas with abiotic stress tolerance. However, studies of the genetic diversity and pattern of genetic structure of M. ruthenica distributed on the Qinghai-Tibetan Plateau (QTP) have been limited. In this study, nuclear internal transcribed spacer (ITS) and chloroplast (psbA-trnH) DNA regions were used to evaluate the genetic diversity, structure and population dynamics of 12 wild M. ruthenica populations distributed on the QTP and Inner Mongolia. Although the chloroplast psbA-trnH region was more variable than nuclear ITS, both markers indicated high interpopulation genetic differentiation, with all populations clustered into two groups based on neighbor joining clustering. Mismatch distribution and neutrality tests revealed local or small-scale demographic expansion. As inferred by morphology and genetic structure, M. ruthenica can be divided into two ecotypes, a result suggested previously. Our findings imply that the genetic divergence of M. ruthenica may be due to climatic oscillations or earlier uplifts of the QTP. (C) 2016 Elsevier Ltd. All rights reserved.