Marine food webs are strongly size-structured and size-based analysis of communities is a useful approach to evaluate food webs in a way that can be compared across systems. Fatty acid analysis is commonly used to identify diet sources of species, offering a powerful complement to stable isotopes, but is rarely applied to size-structured communities. In this study, we used fatty acids and stable isotopes to characterize size-based variation in prey resources and trophic pathways over a nine-month temperate coastal ocean time series of seven plankton size classes, from > 0.7-μm particulate organic matter through > 2000-μm zooplankton. Zooplankton size classes were generally distinguishable by their dietary fatty acids, while stable isotopes revealed more seasonal variability. Fatty acids of zooplankton were correlated with those of their prey (particulate organic matter and smaller zooplankton) and identified trophic pathways, including widespread ties to the microbial food web. Diatom fatty acids also contributed to zooplankton but fall blooms were more important than spring. Concurrent isotope-based trophic position estimates and fatty acid markers of carnivory showed that some indicators (18:1ω9/18:1ω7) are not consistent across size classes, while others (DHA:EPA) are relatively reliable. Both analysis methods provided distinct information to build a more robust understanding of resource use. For example, fatty acid markers showed that trophic position was likely underestimated in 250-μm zooplankton, probably due to their consumption of protists with low isotopic fractionation factors. Applying fatty acid analysis to a size-structured framework provides more insight into trophic pathways than isotopes alone.© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.