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, Anabaena (0–107 cells ml−1) and Microcystis (103–107 cells ml−1) occurred most frequently and dominated in eutrophic systems. A significant linear relationship (n = 31, r2 = 0.38, P < 0.001) between the Microcystis cell numbers and MC concentration (1.3–93 fg of MC cell−1) was observed. Besides [MeAsp3, Mdha7]-MC-RR, two new MCs, [Asp3]-MC-RY and [MeAsp3]-MC-RY, were isolated and their constitution was assigned by LC-MS2. To identify the MC-producing organism in the water samples, (i) the conserved aminotransferase domain part of the mcyE 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 mcyE gene of the genera Microcystis, Anabaena, and Planktothrix. Only mcyE genotypes that are indicative of Microcystis sp. were obtained via the environmental cloning approach (337 bp, 96.1–96.7% similarity to the Microcystis aeruginosa strain PCC7806). Accordingly, only the mcyE primers, which are specific for Microcystis, revealed PCR products. We concluded that Microcystis is the major MC-producer in Ugandan freshwater. © 2009 Wiley Periodicals, Inc. Environ Toxicol 25: 367–380, 2010.