Abstract In this work, we characterised the cyanobacterial communities in the plankton and littoral biofilm of 38 lakes and in the biofilm of 21 rivers in the Alps and surrounding subalpine regions by 16S rRNA gene metabarcoding. We found little overlap in the distribution of amplicon sequence variants (ASVs) between the three habitats and between water bodies. The differences were caused by environmental filtering acting on the selection of the most abundant ASVs and a high contribution of rare oligotypes. The differentiation of community and genotype composition from specific water bodies was explained to a significant extent by environmental variables and morphometry. The taxonomic consistency of ASVs classified under the same genus name was assessed by phylogenetic analyses performed on three representative dominant genera, namely Cyanobium , Tychonema and Planktothrix . The analyses revealed eco-evolutionary adaptations in lakes and rivers, including some evidence for a polyphyletic nature. Monitoring individual genotypes in relation to environmental conditions will be useful to define the ecological amplitude of these taxa. However, the persistence or ephemeral nature of some of the rarest and most unusual ASVs has remained unknown.