The plant Rhodiola crenulata is a perennial herbaceous species distributed in the plateau region of southwestern China, especially the Hengduan Mountains region. It has been one of the most important traditional herbal remedies in Tibet for more than one thousand years, but the accelerated and uncontrolled collection of this plant since the 1980s has lead to deforestation. We used inter-simple sequence repeats (ISSR) to assess levels of genetic variation in R. crenulata from nine diverse natural populations in eastern Tibet and northern Yunnan, the first time such a study has been carried out. The 12 primers we used were able to detect 184 polymorphic loc. Analysis of molecular variance (AMOVA) indicated that species level genetic diversity was relatively high (p = 97.83%, and Ho = 0.464) and analysis using Shannon’s index showed that the within and between genetic diversity of R. crenulata are approximately equal. Nei’s genetic distance and unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis showed that the three populations from Tibet and the six populations from Yunnan form two major clusters. The Yunnan populations from three locations were further divided into three corresponding groups, indicating that genetic differentiation was correlated to geographic distribution. Understanding the genetic structure of R. crenulata provides insight for the conservation and management of this endangered species.