Abstract: 【Aim】 To investigate the genetic differentiation of geographical populations of Luehdorfia chinensis chinensis. 【Methods】 The mitochondrial COI gene and the non-coding fragments of three nuclear genes (ITS2, Pgi-intron and Tpi-intron) of 60 individuals from five geographical populations of L. chinensis chinensis were amplified by PCR. The genetic variation, genetic diversity and genetic differentiation of the five geographical populations were analyzed by using MEGA, DnaSP and Arlequin software, and their demography was inferred. 【Results】 The base sequences of the mitochondrial gene COI and the non-coding fragments of the three nuclear genes ITS2, Pgi-intron and Tpi-intron of the geographical populations of L. chinensis chinensis have strong AT bias. The nucleotide diversity index (π) and the average number of nucleotide differences (K) of the total population were 0.00686 and 18.289, respectively, indicating a high genetic diversity of the total population. The total fixed coefficient (Fst) and the total gene flow (Nm) were 0.4910 and 0.2600, respectively, suggesting that the genetic differentiation among geographical populations is large and the gene communication infrequent. The analysis of molecular variance (AMOVA) showed that the genetic differentiation of L. chinensis chinensis mainly came within a population (52.79%). The Mantel test showed that the geographical distance between populations had a significant positive correlation with the genetic distance (R=0.8519, P<0.05). 【Conclusion】 The five geographical populations of L. chinensis chinensis tested in this study exhibit high genetic diversity, and there are obvious genetic differentiation and little gene communication among the geographical populations. Geographical distance is one of the important factors affecting genetic differentiation among the geographical populations of L. chinensis chinensis. The total population of L. chinensis chinensis has not experienced any recent population expansion.