Rabdosia rubescens (Henmsl.) Hara is an anticancer plant distributed in the hills and mountains of mainland China. In order to evoluate, manage and develop the germplasm genetic resources of this species, 7 natural populations were sampled and analyzed using Inter-simple sequence repeats (ISSR) markers. There was a moderate level of diversity at species level (Ppl: 60.45%; H: 0.2714; I: 0.3365; HB: 0.3108), and a low level of diversity at population level (Ppl: 17.05%; H: 0.0692; I: 0.0989; HB: 0.0763), and a high level of genetic differentiation (GST: 0.6515; θB: 0.6388; GST-B: 0.6095). The Sichuan (SC), Baojing (BJ) and Shimen (SM) populations composed core clade, and then gathered with the southern and northern populations, respectively, based on the UPGMA analysis. Therefore, ISSR markers were suitable for genetic diversity analysis and instructive for core collection construction of R.. rubescens.