Conyza blinii H.Lév.(C. blinii)is a genuine traditional Chinese medicine in Yunnan, Guizhou and Sichuan in China. It has good anti-inflammatory, cough relieving and asthma relieving effects. In order to explore the differences among wild C. blinii populations, nine samples of wild C. blinii from different regions were collected for karyotype, chloroplast genome and phylogeographic structure analysis. The results showed that blinin content, the secondary metabolite of wild C. blinii in Panzhihua area, was higher than that in other areas. C. blinii tenuifolia is diploid. The core formula of cell chromosome 2n = 2x = 20 = 18m + 2st. There is no significant difference in karyotype between populations. Phylogenetic analysis of chloroplast genome showed that Aster spathulifolius was closely related. Based on the cpDNA phylogeographic analysis, it was found that Hap1 was an ancestral type and C. blinii had experienced population expansion, and differences mainly came from within the population. In conclusion, population internal variation and environmental factors may lead to the differences between populations of C. blinii. Our research provides a basis for the development and utilization of wild C. blinii resources.