Understanding the characteristics of trace element enrichment in crayfish from different regions is important for brand protection, quality control, and food safety. Here, we collected farmed and wild crayfish (n = 660) from four regions of China to explore differences in trace element enrichment and find an effective method of identifying their origin. The concentrations of most trace elements in tissue were ranked viscera > exoskeleton > muscle. The concentrations of Co, Ag, Rb, Ba, Se, Cs, and Tl were higher in wild crayfish than in farmed ones. A multi-element analysis shows that there are regional differences in elemental composition. Significant regional differences in delta C-13 and delta N-15 values were also found. The target hazard quotients are all <1, indicating that the normal consumption of crayfish has no significant health risks. This study demonstrates that a combination of multi-element and stable isotope ratio analyses is a practical way to identify the origin of crayfish products.