This resampling study in 338 semi-permanent plots analyses changes in river macrophyte diversity in 70 water courses (small streams to medium-sized rivers) from four regions of the northwest German lowlands during the last six decades. The total macrophyte species pool decreased between the 1950s and 2010/2011 by 28% (from 51 to 37 species), mean plot-level species richness by 19% (from 4.7 to 3.8 species per relevé) and the number of red-listed species by 40% (from 30 to 18 species). Species loss was associated with marked change in species traits: species with presumably higher mechanical stress tolerance (indicated by low specific leaf area and short leaf longevity) are more abundant today. Nearly, half of the species present in the 1950s had either disappeared or been replaced by other species in the recent relevés. The dramatic impoverishment is likely a consequence of continued nutrient input that drove oligo- and mesotraphent species to extinction, and of restructuring and maintenance works in the water courses that reduced stagnant and undisturbed river habitats, where stress-intolerant species can persist. Efficient measures to reduce the nutrient load and to re-naturalise stream and river beds are urgently needed to halt and reverse the loss of macrophyte diversity.