A primary-structure analysis of the 16S rRNA gene was performed with 10 strains representing five described and one unidentified species of the genus Microcystis. The phylogenies determined illustrate the evolutionary affiliations among Microcystis strains, other cyanobacteria, and related plastids and bacteria. A cluster of 10 strains that included hepatotoxic isolates identified as Microcystis aeruginosa formed a monophyletic group. However, the genus Microcystis appeared to be polyphyletic and contained two strains that clustered with unicellular cyanobacteria belonging to the genus Synechococcus. The clustering of related Microcystis strains, including strains involved in the production of the cyclic peptide toxin microcystin, was consistent with cell morphology, gas vacuolation, and the low G + C contents of the genomes. The Microcystis lineage was also distinct from the lineage containing the unicellular genus Synechocystis and the filamentous, heterocyst-forming genus Nostoc. The secondary structure of a Microcystis 16S rRNA molecule was determined, and genus-specific sequence signatures were used to design primers that permitted identification of the potentially toxic cyanobacteria belonging to the genus Microcystis via DNA amplification.