Cardiocrinum giganteum belongs to the genus Cardiocrinum (Endl.) Lindl. (Liliaceae), which is an important wild plant resource. In this study, the genetic diversity and genetic structure of 10 C. giganteum populations were analyzed by using the rpl16 sequence of the chloroplast DNA in order to provide theoretical basis for its resource protection and utilization. By alignment of 699 bp sequence, 14 mutation sites and 12 haplotypes were identified, with the haplotype polymorphism (Hd) and the total genetic diversity (HT) of 0.604 and 0.660, respectively. The genetic variation among the population (71.5 3%) was higher than the intra-population genetic variation (28.47 %), indicating the population variation largely contributed by different populations. The genetic differentiation among the population was high (Gst = 0.677, Nst = 0.614, Fst = 0.71531) and the gene flow was low (Nm = 0.0995). Mismatch analysis revealed that the population had experienced rapid expansion during the evolution process, which was supported by the neutrality tests (Tajima's D = -1.74368 (P > 0.05); Fu and Li's D = -2.69366 (P < 0.05); Fu and Li's F = -2.79896 (P < 0.05)). The study revealed the abundant genetic diversity and differentiation of C. giganteum particularly among populations, which was mainly related to the characteristics of life history. Thus, this work suggested an in-situ conservation strategy being employed in C. giganteum.