Chuanwu (Aconitum carmichaeli Dexb. (Ranunculaceae)) is a traditional Chinese medical herb, widely used for its pharmacological effects, including anti-inflammation and pain relief. This herb has been cultivated in China for medical use for over 1000 years and is mainly grown in Sichuan and Shanxi provinces in China. We have examined the genetic diversity and genetic differentiation in 16 wild populations of A.carmichaeli from HengDuan and QingBa Mountains in Southwest China using random amplified polymorphic DNA (RAPD) markers. A total of 643 discernible loci were obtained for all populations using 24 primers, and 93.5% of these loci were polymorphic (PPL=93.5%). POPOGENE analysis indicated that the genetic diversity by Shannon index among populations was 42.4% and Nei’s gene diversity coefficient (GST) was 40.0%. AMOVA also found the genetic diversity among population was 25.37%. It was found that the genetic variation was relatively greater within populations compared to that among populations. The estimated gene flow from GST(Nm) was 0.756. Wind and insect pollination played important roles in the sexual reproduction of wild A.carmichaeli, resulting in a high level of out-crossing. Out-crossing and tuber reproduction were the main causes of high genetic diversity and abundant genetic differentiation in A.carmichaeli genetic resources. Calculations of Shannon diversity and Nei genetic differentiation consistently indicated that the Chongqing Youyang and Chongqing Chengkou populations possessed the greatest genetic diversity, where as Sichuan Yanyuan and Shanxi Mianxian populations possessed the lowest genetic diversity. The results of UPGMA and Nei’s analysis indicated that the genetic make-up of these wild populations of A.carmichaeli showed regional differences. The populations in a region including Beichuan, Anxian and Qingchuan were quite closely related. The high medical quality variety Jiangyou FUZI (Radix Aconiti Lateralis Preparata) is from this region and the tuber of this variety is usually used as seeds in cultivation. This research has indicated that there is rich germplasm resource of A. carmichaeli and there are large genetic diversities among its wild populations; both are important for selecting A. carmichaeli varieties that may have better medical quality.