Polyphasic analysis was carried out of natural samples and strains isolated from Phormidium mats, collected from different Spanish rivers with distinct water quality. 16S rRNA gene analysis separated five main clusters in a phylogenetic tree corresponding to five main, previously observed morphotypes. Two isolated strains and three natural samples had a relatively homogeneous morphology, which could correspond to Phormidium autumnale, and their sequences grouped in a well-supported cluster (morphotype-genotype I). However, ultrastructural differences were found among representatives from this cluster, and three clades could be separated in a further phylogenetic analysis. The samples belonging to the first clade were characteristic of hypertrophic waters, while representatives of the second grew in mesotrophic conditions, and those of the third in eutrophic waters. These results suggested that our representatives of morphotype-genotype I belong to three different species. According to our results and other published data, complementary information was obtained on the basis of morphological, phylogenetic and ecological criteria. Morphotype-genotype II, corresponding to Phormidium terebriforme, is a potential indicator of polluted waters. Oscillatoria tenuis, corresponding to morphotype-genotype III, is characteristic of eutrophic conditions. Phormidium mats identified as Phormidium corium (morphotype-genotype IV) were found to be characteristic of waters with low nutrient levels. Finally, morphotype-genotype V, identified as Phormidium aerugineo-caeruleum was dominant in oligo-to mesotrophic conditions.