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The composition and abundance of phytoplankton is an important factor defining ecological status of marine and freshwater ecosystems. Chemotaxonomic markers (e.g., pigments and fatty acids) are needed for monitoring changes in a phytoplankton community and to know the nutritional quality of seston for herbivorous zooplankton. Here we investigated the suitability of sterols along with fatty acids as chemotaxonomic markers using multivariate statistics, by analyzing the sterol and fatty acid composition of 10 different phytoplankton classes including altogether 37 strains isolated from freshwater lakes. We were able to detect a total of 47 fatty acids and 29 sterols in our phytoplankton samples, which both differed statistically significantly between phytoplankton classes. Due to the high variation of fatty acid composition among Cyanophyceae, taxonomical differentiation increased when Cyanophyceae were excluded from statistical analysis. Sterol composition was more heterogeneous within class than fatty acids and did not improve separation of phytoplankton classes when used alongside fatty acids. However, we conclude that sterols can provide additional information on the abundance of specific genera within a class which can be generated by using fatty acids. For example, whereas high C-16 omega-3 PUFA (polyunsaturated fatty acid) indicates the presence of Chlorophyceae, a simultaneous high amount of ergosterol could specify the presence of Chlamydomonas spp. (Chlorophyceae). Additionally, we found specific 4 alpha-methyl sterols for distinct Dinophyceae genera, suggesting that 4a-methyl sterols can potentially separate freshwater dinoflagellates from each other.