<jats:title>Abstract</jats:title><jats:p>The high metabolic diversity of potentially toxigenic cyanobacteria has been recently demonstrated for several bloom‐forming genera. To understand the dynamics of cyanobacterial blooms and fluctuations in toxin content, techniques are required that allow the distinction of clones in field samples on a subspecific taxonomic level. We evaluated the possibility to analyze single filaments of a common toxigenic taxon, <jats:italic>Planktothrix rubescens</jats:italic>, by comparing peptide profiles obtained from HPLC/MS analyses of bulk biomass to mass spectra of single filaments for 11 clonal strains. For all strains a significant relationship of chromatogram peak area to mass spectrum peak height was found for individual peptides. Individual peptide's response in mass spectrometry compared to HPLC, however, differed, likely attributable to the presence of particular moieties with high proton affinities in the molecules. Thirty‐four individual peptides detected in HPLC fractions of all strains could also be detected in the corresponding single filaments. In conclusion, MALDI‐TOF MS analysis of single filaments is shown to be an efficient tool for the study of chemotype diversity and dynamics in field studies and laboratory experiments. The good correspondence between peak area and peak height in chromatograms and mass spectra, respectively, is promising with respect to the use of MALDI‐TOF MS as a quantitative analytical tool for natural products studies and cyanotoxin research. Copyright © 2007 John Wiley & Sons, Ltd.