Polyunsaturated fatty acids (PUFA) are crucial nutrients for fish and have been identified as globally-limited nutrients that are needed for ecosystem and human health. Greater understanding is needed into the processes influencing observed PUFA levels in fish as human demand for PUFAs increases because of their nutritional benefits and anthropogenic stresses threaten to reduce PUFA production. We present a general, process-based mass balance model for freshwater fish that estimates concentrations of n-3 (α-linolenic acid: ALA, eicosapentaenoic acid: EPA and docosahexaenoic acid: DHA) and n-6 (linoleic acid: LIN and arachidonic acid: ARA) PUFA from prey food items. Our model considers the processes of dietary uptake, absorption efficiency, egestion, transformation (elongation and/or desaturation) and β-oxidation. The model relies on rate constants derived from multiple regression analysis for egestion, transformation and β-oxidation based on ecological and physiological variables (i.e. body weight, diet, PUFA interactions). All regression equations had adjusted R2≥0.47 and p values<0.001. Application of the model to Yellow Perch (Perca flavescens) from the Upper Bay of Quinte, Canada provided estimates of ALA, EPA, and DHA, LIN and ARA contents that were within a standard deviation of measured values without model calibration. The model showed that diet was the main source of ALA, EPA, LIN and ARA. Transformation of EPA to DHA was the dominant source of DHA. We hypothesize that within-fish transformation of precursor and the resultant product PUFA can compensate, to some extent, for dietary deficiencies in long chain PUFA in the diet of this freshwater fish.