Cyanobacteria can form extensive blooms in water with concurrent production and release of a large number of chemically diverse and bioactive metabolites, including hazardous toxins. Significant number of the metabolites belongs to non-ribosomal peptides, with unique residues, unusual structures and great potential for biotechnological application. The biosynthetic pathways of the peptides generate tens of variants, but only part of them has been identified. Microginins are an understudied class of cyanobacterial linear peptides with a characteristic decanoic acid derivative amino acid residue in their structure. In this study, cyanobacterial blooms and isolated strains from Greek lakes were analyzed for the presence of microginins by liquid chromatography coupled to hybrid triple quadrupole/linear ion trap mass spectrometer (LC-qTRAP MS/MS). Microginin structures were elucidated based on the obtained fragmentation spectra. A large number of microginins occurred in blooms of Greek freshwaters and the most frequently detected were Microginin FR1 (70% of samples), Microginin T1 (52%), Microginin 565B (52%), Microginin T2 (43%), and Microginin 565A (43%). Additionally, nine cyanobacterial strains i.e. Nostoc oryzae, Synechococcus sp., Microcystis aeruginosa, Microcystis viridis, and five Microcystis sp., were found to produce microginins. Thirty-six new microginin structures were characterized out of fifty-one totally detected variants. This is the first time that such a diversity of microginins is reported to be present in water bodies. Results clearly demonstrate the great metabolomic potential of cyanobacteria that inhabit Greek freshwaters and significantly expand the knowledge of cyanobacterial secondary metabolites with regards to the class of microginins.