The role of within-species genetic variability and ecotypes has been poorly documented in research on water bioindication. Some studies already show that macrophytes could present genetic lineages with different optima for specific water parameters. In the present study, we tested this assumption on Potamogeton coloratus Hornem., a macrophyte described as characteristic of oligotrophic waters and threatened by freshwater eutrophication. Species population genetics, water parameters, and macrophyte and macroinvertebrate communities were analysed in 29 sites among three eco-regions of the eastern parts of France: Rhine Plain, Rhône Valley and Provence. The observed diversity of habitats can be divided into two main categories: the lotic hydrosystems such as springs and small watercourses often fed by groundwater, and the lentic waterbodies such as shallow pools or pond sides. Sampling sites were characterised by alkaline (mean pH 7.5 ± 0.4) and cool waters (mean 15.8 ± 4.1 °C) with low N-NH4+ concentration (median value of 20 μg/L), but large variation in other parameters (e.g. nitrates, phosphates). We found two main AFLP genotypic lineages, despite a poor genetic variability mainly distributed between populations, without regional structuring nor within-population diversity. We also sequenced only two ribotypes based on the nrDNA ITS. Genotypes, ribotypes and habitat categories were not correlated, arguing that the divergence of genotypic lineages is not due to ecotypic differentiation for P. coloratus. We highlight a putative event of long-distance dispersal, which illustrates the species ability for recolonization. Finally, we provide some suggestions for its conservation facing global changes.