The fatty acid (FA) composition of algae is an important determinant of their food quality for consumers, and FAs can also be used as biomarkers for biochemical and energetic pathways in food webs. FA analyses of 7 freshwater algal classes and 37 strains showed clear similarity within classes and strong differences amongst classes. Class was a dominant factor (66.4%) explaining variation in FA signatures of microalgae. The 7 algal classes comprised 4 separate groups according to their FA profiles: (1) Chlorophyceae and Trebouxiophyceae, (2) Bacillariophyceae, (3) Cryptophyceae, Chrysophyceae, and Raphidophyceae, and (4) Euglenophyceae. Each group had a characteristic FA composition, although the proportional abundance of individual FAs also differed between species and with environmental conditions. FAs found to be particularly representative for each group (i.e. diagnostic biomarkers) were as follows: 16:4ω3 and 16:3ω3 for Chlorophyceae and Trebouxiophyceae; 16:2ω7, 16:2ω4, 16:3ω4, 16:4ω1, and 18:4ω4 for Bacillariophyceae; 22:5ω6 and 18:4ω3 for Cryptophyceae and Chrysophyceae (Synurales), 16:3ω1 for Chrysophyceae (Ochromonadales), 16:2ω4, 16:3ω4, 16:3ω1, and 20:3ω3 for Raphidophyceae; and 15:4ω2, 20:4ω3, 20:2ω6, 20:3ω6, and 22:4ω6 for Euglenophyceae. FAs thus offer a powerful tool to track different consumer diets in a lacustrine food web. Based on the 20:5ω3 (eicosapentaenoic acid) and 22:6ω3 (docosahexaenoic acid) content among the investigated freshwater algal classes, Chlorophyceae, Trebouxiophyceae, and Chrysophyceae are of intermediate food quality for zooplankton, and Cryptophyceae, Bacillariophyceae, Euglenophyceae, and Raphidophyceae should be excellent resources for zooplankton.