The objective of the research described in this paper was to evaluate the level of genetic variability and differentiation between the populations of Hedysarum theinum Krasnob., endemic species of Altai-Sayan Mountains. In this study, six inter-simple sequence repeat (ISSR) primers were used and generated a total of 132 molecular markers, 126 of which were polymorphic (95.5%). The large amount ofDNA polymorphism at the intrapopulation level (H(pop)/H(sP) = 70.5) was evinced by the analysis of a molecular variance (AMOVA) of 88.2%. The high genetic similarity (I = 0.875) detected between the populations of H. theinum reflects the strongly restricted endemic range of H. theinum, which facilitates the gene flow among populations and out-crossing. Due to the significant genetic diversity revealed among individuals against a background of moderate population differentiation collecting samples for ex situ H. theinum conservation from a small number of populations is acceptable. Finally, selected highly polymorphic ISSR markers produced consistently repeatable patterns are discussed as a powerful tool for genotype identification and the qualification of root feedstock.