Abstract Suspended matter from surface microlayers and various water depths, plankton and surface sediments have been collected at various Mediterranean sites and have been subjected to flash pyrolysis techniques to study the scope of pyrolysis methods in the analysis of the chemical composition of suspended organic matter in the ocean. Samples have been analysed by both pyrolysis-mass spectrometry (Py-MS) and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). Discriminant analysis of the Py-MS spectra of 26 samples indicated the amplitude and nature of the variance between the samples. By means of Py-MS fingerprints, the samples are separated into well-defined clusters, reflecting the nature, the geographic origin and the sampling depth. The differences observed are due to varying amounts of peptides, polysaccharides and lipids. The plankton samples are clearly separated due to their high content in recently biosynthesized material, such as proteins. Other samples collected in coastal areas, especially in the Northern Adriatic Sea, are characterized by relatively high contents of polysaccharides probably due to high riverine nutrient inputs and consequently a high plankton productivity. The chemical interpretations of the Py-MS data are based on Py-GC-MS data of a few selected samples. Py-GC-MS traces allow the detailed identification of pyrolysis products derived from peptides, polysaccharides, lipids, lignin and others. Flash pyrolysis techniques appear to be a useful tool to study the chemical nature of suspended matter, allowing the determination of major building blocks of complex mixtures of biopolymers, degraded biopolymers and condensation products, present in the suspended matter of the ocean.