Conyza blinii H.Lév. (C. blinii) is a plant species with natural wild distribution in Yunnan, Guizhou and Sichuan in China. It’s considered as genuine traditional Chinese medicine, since it has good anti-inflammatory, cough relieving and asthma relieving effects. In order to explore the diversity among wild C. blinii populations, nine samples collected from different regions were analyzed for karyotype, chloroplast genome and phylogeographic structure. The results showed that blinin content, a secondary metabolite of wild C. blinii, was higher in Panzhihua area than that in other areas. C. blinii tenuifolia was identified as diploid species (2n = 2x = 20 = 18m + 2st), and no significant difference in karyotype between populations was detected. Phylogenetic analysis of chloroplast genome showed a close relation with Aster spathulifolius. The cpDNA phylogeographic analysis revealed that Hap1 was an ancestral type. The population expansion in C. blinii was detected, and the genetic diversity was largely contributed by intra-population variations. Collectively, the intra-population variation and environmental factors may lead to the differentiation between populations of C. blinii, thus providing insights for future development and utilization of wild C. blinii resources.