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<i>Rheum tanguticum</i> is a widely used Chinese medicinal plant. Recently, because of the great demand, the wild populations have been declining rapidly. In this study, the levels of genetic variation of 11 wild and five cultivated populations of <i>R. tanguticum</i> were investigated by ISSR markers. The 13 selected ISSR primers amplified 306 polymorphic bands out of a total of 326 (93.87 %). Based on Nei’s gene diversity and Shannon’s index, the genetic diversity in cultivated populations of <i>R. tanguticum</i> (<i>H</i> = 0.2490; <i>I</i> = 0.3812; <i>H</i> <sub>B</sub> = 0.3033) was relatively lower than that of wild populations (<i>H</i> = 0.2666; <i>I</i> = 0.4124; <i>H</i> <sub>B</sub> = 0.3115), although no significant differences were identified. Assignment was performed with AFLPOP program, and XGM was the most likely source population of HM. The origins of the rest cultivated populations were admixture. UPGMA and PCoA analyses showed that wild and cultivated populations were not separated into two groups, indicating that a large number of wild genotypes were maintained in the cultivated gene pool. The coefficient of genetic differentiation between wild and cultivated populations was 0.0305 (<i>G</i> <sub>st</sub>), which was in good agreement with the results of analysis of molecular variance (AMOVA), in which, only 1.85 % of the total variance existed between groups of wild and cultivated populations, while 70.91 % occurred within populations and 27.24 % among populations. Together, these results indicated that cultivated populations were not genetically differentiated from wild populations. On the basis of this study, we have made some suggestions for the conservation and efficient management of the genetic resources of this important medicinal herb.