Abstract: We used the second intron of the LEAFY gene to analyze the genetic diversity of Isoetes sinensis Palmer from China. In total, 105 samples were collected from seven populations. From these I. sinensis samples, 78 haplotypes were discovered, with a haplotype diversity of 0.989 and nucleotide diversity of 0.021. Results showed that genetic differentiation coefficient G_ST=0.283, N_ST=0.298, G_ST>N_ST, N_m=0.59; Mantel test, Rxy=-0.286, P (rxy-rand≥rxy-data)=0.370, and 72% of the total variation existed within populations. UPGMA cluster showed that the two high altitude populations had distant genetic relationships with the other five populations. The neutrality test, Tajima's D, Fu & Li's D^* and F^* tests were negative, and the mismatch distribution test based on a stable model was multimodal. According to the geographic location of I. sinensis, its genetic structure was related to the drainage, altitude, and process of hybrid polyploidization.