Interpopulation gene flow plays an important role in determining population genetic structure. In this study, we sampled 24 populations of the whorl-leaf watermilfoil Myriophyllum verticillatum from three disjunct regions in China (Northwest China, Northeast China, and the Qinghai-Tibet Plateau (QTP) plus Southwest China) with different topographies and examined its population genetic structure using 17 microsatellite loci. Deserts, plains, and mountains are the main geomorphological features of Northwest China, Northeast China, and the QTP plus Southwest China, respectively. High genetic differentiation and genetic isolation by geographic distance were observed at the overall scale and in all three regions. When compared among the three regions, the lowest level of genetic differentiation and weakest correlation between genetic and geographic distances were presented in Northeast China. Two genetic clusters corresponding to Northwest China and the other two regions were identified, suggesting unequal gene flow among the three disjunct regions. Our findings emphasize the role of topography and geographic distance in shaping population genetic structure in aquatic plants.