Omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) are essential micronutrients for aquatic consumers. Synthesized by aquatic primary producers, n-3 LC-PUFA are transferred across trophic levels and may eventually end up accumulating in fish. However, if short in dietary supply, fish may also biosynthesize n-3 LC-PUFA from dietary precursors (i.e., n-3 C18-PUFA). We applied compound-specific hydrogen stable isotope analysis (CSIA) of fatty acids to investigate sources and metabolic processes of n-3 LC-PUFA, and in particular of docosahexaenoic acid (22:6n-3, DHA), in Common Carp (Cyprinus carpio) raised in semi-intensive aquaculture ponds. Carp were feeding on natural pond zooplankton and benthic macroinvertebrates rich in n-3 LC-PUFA and cereal-based pellet feeds rich in C18-PUFA. Results provide isotopic evidence that carp obtained a significant amount of dietary lipids and nitrogen from added cereal-based feeds, while n-3 LC-PUFA were generally acquired by feeding on benthic macroinvertebrates and zooplankton. However, DHA retained in carp was also generated endogenously via bioconversion from dietary PUFA precursors, such as EPA. DHA was isotopically lighter than EPA and likely not supplied in sufficient quantities to meet the physiological requirements for DHA in carp. Our data show that depending on the natural abundance of dietary DHA in these eutrophic ponds, farmed carp can obtain DHA by two different pathways; i.e., directly via dietary uptake and indirectly via bioconversion. This field study highlights the importance of dietary LC-PUFA supply in eutrophic aquatic ecosystems and the ability of carp to biosynthesize highly valuable LC-PUFA, eventually also benefiting human health.