Abstract: Eleven species of Anemone obtusiloba collected from 4 areas of Gansu were analysed by inter single sequence repeats (ISSR) to elucidate the inherit diversity level and structure. Seven hundred and one bands were amplified by 13 primers and 392 bands were polymorphic, a polymorphic locus percentage (P) of 55.92%. The Shannon index (I) was 0.372 and Nei’s gene diversity (H) was 0.250. Based on Nei’s Gst value and analysis of molecular variance (AMOVA), showed that species inheritance of differentiation of A. obtusiloba existed mainly. In inter-populations: I was 0.183 and H was 0.114 at the population level. These data indicated that there were high inherent diversities in the population of A. obtusiloba. Using UPGMA, three populations were clustered: HZ, LQ and AWC, and AZ. Further analysis showed that there was no correlation between population genetic diversity level and altitude, water, extractable phosphorous or longitude and latitude, but there was a significantly negative correlation with precipitation.