<jats:p>In recent years, the occurrence of cyanobacterial blooms in eutrophic freshwaters has been described all over the world, including most European countries. Blooms of cyanobacteria may produce mixtures of toxic secondary metabolites, called cyanotoxins. Among these, the most studied are microcystins, a group of cyclic heptapeptides, because of their potent hepatotoxicity and activity as tumour promoters. Other peptide cyanotoxins have been described whose structure and toxicity have not been thoroughly studied.<jats:p>Herein we present a peptidomic approach aimed to characterise and quantify the peptide cyanotoxins produced in two Italian lakes, Averno and Albano. The procedure was based on matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry mass spectrometry (MALDI‐TOF‐MS) analysis for rapid detection and profiling of the peptide mixture complexity, combined with liquid chromatography/electrospray ionisation quadrupole time‐of‐ flight tandem mass spectrometry (LC/ESI‐Q‐TOF‐MS/MS) which provided unambiguous structural identification of the main compounds, as well as accurate quantitative analysis of microcystins. In the case of Lake Averno, a novel variant of microcystin‐RR and two novel anabaenopeptin variants (Anabaenopeptins B<jats:sub>1</jats:sub> and Anabaenopeptin F<jats:sub>1</jats:sub>), presenting homoarginine in place of the commonly found arginine, were detected and characterised. In Lake Albano, the peculiar peptide patterns in different years were compared, as an example of the potentiality of the peptidomic approach for fast screening analysis, prior to fine structural analysis and determination of cyanotoxins, which included six novel aeruginosin variants. This approach allows for wide range monitoring of cyanobacteria blooms, and to collect data for evaluating possible health risks to consumers, through the panel of the compounds produced along different years. Copyright © 2011 John Wiley & Sons, Ltd.