<jats:title>Abstract</jats:title><jats:p>Microcystins (MCs) are cyclic heptapeptides, which are the most abundant toxins produced by cyanobacteria in freshwater. The phytoplankton of many freshwater lakes in Eastern Africa is dominated by cyanobacteria. Less is known, however, on the occurrence of MC producers and the production of MCs. Twelve Ugandan freshwater habitats ranging from mesotrophic to hypertrophic conditions were sampled in May and June of 2004 and April of 2008 and were analyzed for their physicochemical parameters, phytoplankton composition, and MC concentrations. Among the group of the potential MC‐producing cyanobacteria, <jats:italic>Anabaena</jats:italic> (0–10<jats:sup>7</jats:sup> cells ml<jats:sup>−1</jats:sup>) and <jats:italic>Microcystis</jats:italic> (10<jats:sup>3</jats:sup>–10<jats:sup>7</jats:sup> cells ml<jats:sup>−1</jats:sup>) occurred most frequently and dominated in eutrophic systems. A significant linear relationship (<jats:italic>n</jats:italic> = 31, <jats:italic>r</jats:italic><jats:sup>2</jats:sup> = 0.38, <jats:italic>P</jats:italic> < 0.001) between the <jats:italic>Microcystis</jats:italic> cell numbers and MC concentration (1.3–93 fg of MC cell<jats:sup>−1</jats:sup>) was observed. Besides [MeAsp<jats:sup>3</jats:sup>, Mdha<jats:sup>7</jats:sup>]‐MC‐RR, two new MCs, [Asp<jats:sup>3</jats:sup>]‐MC‐RY and [MeAsp<jats:sup>3</jats:sup>]‐MC‐RY, were isolated and their constitution was assigned by LC‐MS<jats:sup>2</jats:sup>. To identify the MC‐producing organism in the water samples, (i) the conserved aminotransferase domain part of the <jats:italic>mcy</jats:italic>E gene that is indicative of MC production was amplified by general primers and cloned and sequenced, and (ii) genus‐specific primers were used to amplify the <jats:italic>mcy</jats:italic>E gene of the genera <jats:italic>Microcystis</jats:italic>, <jats:italic>Anabaena</jats:italic>, and <jats:italic>Planktothrix</jats:italic>. Only <jats:italic>mcy</jats:italic>E genotypes that are indicative of <jats:italic>Microcystis</jats:italic> sp. were obtained via the environmental cloning approach (337 bp, 96.1–96.7% similarity to the <jats:italic>Microcystis aeruginosa</jats:italic> strain PCC7806). Accordingly, only the <jats:italic>mcy</jats:italic>E primers, which are specific for <jats:italic>Microcystis</jats:italic>, revealed PCR products. We concluded that <jats:italic>Microcystis</jats:italic> is the major MC‐producer in Ugandan freshwater. © 2009 Wiley Periodicals, Inc. Environ Toxicol 25: 367–380, 2010.