Resource polymorphism—whereby ancestral generalist populations give rise to several specialised morphs along a resource gradient—is common where species colonise newly formed ecosystems. This phenomenon is particularly well documented in freshwater fish populations inhabiting postglacial lakes formed at the end of the last ice age. However, knowledge on how such differential exploitation of resources across contrasting habitats might be reflected in the biochemical compositions of diverging populations is still limited, though such patterns might be expected. Here, we aimed to assess how fatty acids (FA)—an important biochemical component of animal tissues—diverged across a polymorphic complex of European whitefish (Coregonus lavaretus) and their closely related monomorphic specialist congener vendace (Coregonus albula) inhabiting a series of six subarctic lakes in northern Fennoscandia. We also explored patterns of FA composition in whitefish’s predators and invertebrate prey to assess how divergence in trophic ecology between whitefish morphs would relate to biochemical profiles of their key food web associates. Lastly, we assessed how information on trophic divergence provided by differential FA composition compared to evidence of resource polymorphism retrieved from more classical stomach content and stable isotopic (δ13C, δ15N) information. Examination of stomach contents provided high-resolution information on recently consumed prey, whereas stable isotopes indicated broad-scale patterns of benthic-pelagic resource use differentiation at different trophic levels. Linear discriminant analysis based on FA composition was substantially more successful in identifying whitefish morphs and their congener vendace as distinct groupings when compared to the other two methods. Three major FA (myristic acid, stearic acid, and eicosadienoic acid) proved particularly informative, both in delineating coregonid groups, and identifying patterns of pelagic-benthic feeding throughout the wider food web. Myristic acid (14:0) content and δ13C ratios in muscle tissue were positively correlated across fish taxa, and together provided the clearest segregation of fishes exploiting contrasting pelagic and benthic niches. In general, our findings highlight the potential of FA analysis for identifying resource polymorphism in animal populations where this phenomenon occurs, and suggest that this technique may provide greater resolution than more traditional methods typically used for this purpose.