Abstract: Water flow plays a crucial role in the propagation of aquatic invasive plants. Eichhornia crassipes is a major invasive aquatic plant in China, causing significant harm. Using AFLP markers, this study explored the clonal diversity and genetic structure of 14 E. crassipes populations (comprising 399 individuals) from the middle and lower reaches of the Yangtze River. Based on four pairs of AFLP primers, 186 loci were identified throughout all populations, including 56 polymorphic loci (30.11%). The populations exhibited low clonal diversity (D = 0.273) and low genetic diversity (I = 0.011, H_E = 0.017), due to the dominant clonal reproduction system of the species. Genetic differentiation among populations was minimal (G_ST = 0.019). Analysis of molecular variance (AMOVA) indicated that 1.84% of the genetic variation was attributed to differences among populations, while 98.16% originated from within populations. Additionally, one common genotype was shared by all 14 populations. This study highlighted the significant role of water flow as a primary medium for the dispersal of E. crassipes in the Yangtze River Basin. Therefore, controlling the dispersal of water flow would be a crucial measure for effectively managing the spread of this species.