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1. Previously, the Yangtze River connected thousands of shallow lakes which together formed a potamo-lacustrine system capable of sustaining a rich variety of submerged macrophytes. 2. Potamogeton malaianus is one of the dominant submerged macrophytes in many lakes of this area. Genetic variation and population structure of P. malaianus populations from ten lakes in the potamo-lacustrine system were assessed using inter-simple sequence repeat markers. 3. Twelve primer combinations produced a total of 166 unambiguous bands of which 117 (70.5%) were polymorphic. Potamogeton malaianus exhibited a moderate level of population genetic diversity ( PP = 70.5%, HE = 0.163 and I = 0.255), as compared with that of plants in the same habitat and range. The main factors responsible for this moderate value were the plant’s mixed breeding system (both sexual and asexual) and the hydrological connectivity among habitats. 4. F statistics, calculated using different approaches, consistently revealed a moderate genetic differentiation among populations, contributing about 20% of total genetic diversity. An estimate of gene flow (using FST) suggested that gene flow played a more important role than genetic drift in the current population genetic structure of P. malaianus ( Nm = 1.131). 5. The genetic diversity of P. malaianus did not increase downstream. A high level of linkage–disequilibrium at the whole population level suggested that metapopulation processes may affect genetic structure. The migration pattern of P. malaianus was best explained by a two-dimensional stepping stone model, indicating that bird-mediated dispersal could greatly influence gene movements among lakes. [ABSTRACT FROM AUTHOR]