Abstract: Microcystis blooms, increasingly frequent in lakes, reservoirs, and other aquatic ecosystems, raise concerns due to microcystins (MCs) production. Microcystis wesenbergii, one of the predominant bloom-forming species, has traditionally been considered a non-toxic strain. In this study, immunofluorescence analysis revealed positive reactivity of a purified M. wesenbergii strain (Hui-8) to the microcystin synthetase protein McyF antibody, while molecular characterization confirmed its taxonomic identity as M. wesenbergii and revealed toxin-producing genes (mcyB and mcyD). Quantitative ELISA analysis indicated that the toxin production capacity of Hui-8 was lower than that of the toxic reference strain M. aeruginosa PCC 7806. Subsequent HPLC-MS analysis identified the toxin variant as MC-RR. MCs exposure impair chironomid larvae mobility, gut microbiota, and lipid metabolism; disrupt calcium and potassium homeostasis in submerged plants, causing osmotic stress and leaf damage; and induce rapid cell deformation, inactivation, or necrosis via PP1/PP2A inhibition, leading to cytoskeleton disintegration and organelle dysfunction with prolonged exposure. Most Microcystis species produce MCs, with M. aeruginosa extensively studied for high MC production. For M. wesenbergii, limited evidence—single toxin gene detection and animal experiments—leaves its toxicity inconclusive, and no toxin genes are reported in its genomic data, reinforcing its non-toxic label. However, using McyF nanobodies targeting toxin synthesis gene, some M. wesenbergii strains showed positive results. This study analyzed laboratory-purified M. wesenbergii strains and identified one strain capable of producing microcystins. The colonial M. wesenbergii strains (Hui-8) used in this study were isolated, purified, and maintained under laboratory conditions with stable morphology for three years. Based on the typical M. wesenbergii strain (Hui-8), we compared its physiological differences with non-toxigenic M. wesenbergii and toxigenic M. aeruginosa. Through immunofluorescence localization, PCR detection of toxin genes (mcyB/mcyD), and ELISA quantification confirmed that M. wesenbergii (Hui-8) synthesizes microcystin-RR (MC-RR), challenging the traditional classification of this species as non-toxigenic. Using the McyF nanobody F4-EGFP, Hui-8 bound significantly to the fluorescent nanobody—similar to toxic M. aeruginosa PCC7806—unlike colonial M. wesenbergii TH29 and unicellular FACHB-929suggesting potential toxicity. PCR confirmed mcyB and mcyD in Hui-8, with sequences matching GenBank records. Notably, while the McyF nanobody test was positive, PCR failed to amplify mcyF, mcyA, and mcyC, possibly due to sequence divergence from M. aeruginosa-derived primers or atypical gene organization in M. wesenbergii. PCR detection of mcyB and mcyD does not necessarily confirm toxin production. Therefore, an MCs ELISA kit was used. The results showed that Hui-8has a certain toxin-producing capability, with a toxin concentration of approximately 1.44×10^–2 ng/mg, which is significantly lower than that of M. aeruginosa PCC7806 (6.8×10^–2 ng/mg). HPLC-MS identified a peak at 2.3min matching MC-RR standards, with another peak at approximately 5.2min suggesting a possible uncharacterized toxin variant.