Inferences about ecological structure and function are often made using elemental or macromolecular tracers of food web structure. For example, inferences about food chain length are often made using stable isotope ratios of top predators and consumer food sources are often inferred from both stable isotopes and fatty acid (FA) content in consumer tissues. The use of FAs as tracers implies some degree of macromolecular conservation across trophic interactions, but many FAs are critically important for particular physiological functions and animals may selectively retain or extract these critical FAs from food resources. Here, we compared spatial variation in two taxa that feed on the same (or similar) food resources to assess which FAs appear to be responding to a common gradient in food resources. Filter feeding caddisflies (Family Hydropyschidae) and dreissenid mussels (Genus Dreissena) both consume seston, and had similar spatial variation in stable isotopes (C and N) across 13 sites in the Great Lakes region of North America. Only one of forty-one FAs measured showed strong spatial co-variance in these taxa (α-linolenic acid; ALA), indicating other FAs are responding to other environmental gradients in at least one of these taxa. Based on other experimental studies, ALA does appear to be driven by food availability in caddisflies, so it seems likely that ALA spatial co-variance reflects spatial variation in this food resource in this study. We conclude that inferences made using FAs as tracers of food web structure may be very sensitive to the individual taxa studied.