Wild populations of Nelumbo nucifera are valuable genetic resources and provide significant ecological functions for aquatic ecosystems. However, they have suffered from habitat fragmentation and degradation. Here, the genetic connectivity and variation of N. nucifera wild populations were analysed based on 22 microsatellite markers and 10 populations along the Yangtze River. Abundant historical gene flows were found among populations (Nm: 1.806–3.735). In contrast, low levels of recent migration rates were revealed and only two migrants were detected in the first generation among populations. The results indicated that habitat deteriorations have resulted in dramatically reduced gene flow, and the seasonal floods failed to act as a valid path for migration. The bidirectional gene flows, combined with the lack of accumulation effects in downstream populations, suggested that both river flow and zoochory (or human activity) contributed to gene exchange. High levels of genetic differentiation were found in all pairs of populations (FST: 0.208 - 0.666; G”ST: 0.306 - 0.866) and in the global estimate across all populations (FST=0.403, G”ST = 0.593), indicating genetic discontinuities among populations. The genetic depauperation (HE = 0.273) and obvious genetic structure in N. nucifera populations were explained by reduced gene flow and accentuated genetic drift, combined with the inbreeding and repeated genetic bottlenecks. Based on these results, a conservation strategy for protecting all isolated populations to maintain genetic diversity of wild lotus is proposed, and hydrological connectivity should be facilitated across the aquatic system.