This study examined a range of diets and two tissue types (muscle and carapace, representing protein and chitin biochemistry respectively) of Cherax destructor (Clark, 1936) to allow more accurate use of isotope data in trophic source estimates. The resulting Δ13Ctissue–diet and Δ15Ntissue–diet discrimination factors of muscle and carapace tissues showed significant differences among diets. For muscle, Δ13Ctissue–diet was higher (2.11–2.33‰) when C. destructor was fed with lamb, turkey and mixed animal and plant-based diets, 1.27–1.96‰ when C. destructor was fed with beef and kangaroo diets and negative (–1.36‰) when C. destructor was fed with an aquatic meat (tuna) diet. The Δ15Ntissue–diet discrimination factors were lower for muscle when C. destructor was fed aquatic meat (0.12‰) and mixed plant–animal diets (1.67‰), but higher for terrestrial meat diets (2.79–3.74‰). The Δ13Ctissue–diet for carapace followed similar patterns to that of muscle, but Δ15Ntissue–diet values were lower for carapace than muscle. Strong correlations were observed between muscle and carapace for δ13C (r = 0.96, P < 0.0001) and δ15N (r = 0.82, P < 0.0012) across the six diets evaluated, indicating that carapace can be used as a non-lethal alternative to muscle during field sampling.