Abstract: The genetic characteristics of Hydrilla verticillata (L.f.) Royle were analyzed with chloroplast DNA (cpDNA) markers. Out of 18 candidate pairs, three effective molecular markers (trnL-trnF, trnH-psbA, RPL16) were screened, providing the molecular basis for further research. In total, 12 haplotypes were discovered in 40 samples of H. verticillata, with 135 mutation sites and haplotype diversity of 0.9064. Haplotype-4 was found in regions SXHZ and YY, which were geographically distant. In addition, 85.79% of total variation existed between populations. Through asexual reproduction, H. verticillata could carry out rapid expansion. Geographical separation and various environments between populations have likely resulted in low gene flow. In addition, the genetic structure of H. verticillata has been profoundly affected by founder effect and genetic drift.