Stable isotopes of lake waters are widely used to identify the relative importance of hydrological processes on the lake water balance across the ungauged landscape via the coupled-isotope tracer model. The isotopic compositions of twenty shallow freshwater lakes across the mid-lower reaches of Yangtze floodplain (MLY) were investigated in January and May of 2018. The lake-specific input water (δI) and evaporation-to-inflow (E/I) ratios were estimated to explore the specific lake hydrology across the MLY. Results showed that distinct isotopic enrichment trends in May compared with those in January, which was indicative of stronger evaporation in May. The δ18OI values of specific lakes exhibited large variability across the MLY, which may be related to the watershed properties, such as watershed area and elevation, and rainfall. The estimated E/I ratios of lakes across the MLY were below 1, which suggested that these lakes (code 1–15) are flood-dominated in the middle reaches of Yangtze River where lakes are susceptible to Three Gorges Dams regulations. By contrast, the relatively lower variability of lake E/I ratios were observed from the Yangtze River Delta (code 17–20) because these lakes with developed river network systems are highly exchanged by artificial regulation. Our investigation of lake types and corresponding isotopic evolution patterns are likely typical of other floodplain landscapes and their identification could be used to better predict hydrological responses to ongoing climate change and artificial regulations by dams.