Abstract Organisms at the base of stream food webs are typically poor in long‐chain polyunsaturated fatty acids (LC‐PUFA), especially in docosahexaenoic acid (DHA), whereas consumers at higher trophic levels are often rich in LC‐PUFA. For example, fish tissues, especially the brain, are DHA‐rich. This obvious mismatch between consumer LC‐PUFA and their basal dietary supply may result from selective retention and/or endogenous conversion of dietary precursors to LC‐PUFA. To determine which is more likely, we investigated compound‐specific carbon stable isotopes in PUFA (δ¹³C_PUFA) of potential basal resources (stream epilithon, leaf litter) and consumers (invertebrates, European bullhead, and two salmonid species and their brain, eye, liver, and muscle tissues). We predicted that consumer‐PUFA, depleted in ¹³C values relative to their dietary sources, would indicate internal de novo PUFA synthesis. Alternatively, higher consumer‐δ¹³C_PUFA values would imply selective retention of aquatic (epilithon and conditioned leaves) rather than terrestrial resources or internal production, irrespective of trophic levels. Invertebrate grazers and predators resembled δ¹³C values of essential fatty acids (δ¹³C_EFA) of benthic algae, while shredder‐δ¹³C_EFA values reflected those of conditioned rather than fresh leaves. Lower eye‐δ¹³C_EFA values of salmonids than in livers indicated high retention of dietary PUFA sources (invertebrates and epilithon). Stable isotope values of eicosapentaenoic acid suggest that all consumers retained algal EPA, while insectivorous fish produced DHA in their liver. There is no further evidence from carbon stable isotopes for local PUFA conversion within neural tissues. Our study demonstrates that δ¹³C_PUFA can be used to track sources of these highly functional molecules in aquatic consumers and highlights the importance of algal derived‐PUFA for these consumers in oligotrophic headwater streams.