Malus sieversii constitute a valuable genetic resource in wild apple ecosystems. The aim of this study was to use inter-simple sequence repeat (ISSR) primers as an accessible tool to investigate the genetic diversity in Malus sieversii species. The experimental materials include 34 samples from Kyrgyzstan and Xinjiang of China. A total of 125 bands and 98 polymorphic bands were amplified using 47 ISSR primers. The polymorphism rate was 78.4%. The genetic similarity coefficient of Kyrgyzstan and Xinjiang of China population was 0.68; the genetic similarity coefficient of various populations in Xinjiang was 0.72~0.94. The samples in same population got into a category, but some samples in faraway geographic locations have cross clustering. Geographical isolation hindered the gene exchange of Malus sieversii in different populations for a long time, and Malus sieversii developed along the natural selection environment direction and generate genetic differentiation after that.