Divergence in habitat use among closely related species is a common characteristic of adaptive radiations. Large differences in the size structure of prey between habitats could strengthen disruptive selection on generalist predators and lead to a divergence in trophic position among species in an adaptive radiation. Using threespine stickleback (Gasterosteus aculeatus) in freshwater lakes as a model system, we examined whether divergence in habitat use coincides with shifts in trophic position. We examined the habitat use and trophic position of individual sticklebacks from divergent lake environments that have only one stickleback species (allopatric lakes) and from lakes that have a pair of benthic and limnetic stickleback species (sympatric lakes). In two sympatric lakes, the limnetic species had a higher trophic position than the benthic species, and in both allopatric and sympatric lakes, sticklebacks specializing on pelagic prey had a higher trophic position for a given size than sticklebacks specializing on benthic prey. Furthermore, the trophic position of pelagic specialists was correlated with individual variation in their gill raker length. Our results indicate that gill raker length is an important trait that underlies differentiation in both habitat use and trophic position among stickleback species, populations, and individuals.