Stable carbon isotope ratios can be used to discriminate between wild and sterile insects that are caught in surveillance traps when 2 isotopically distinct dietary sources are available for the immature or adult stages. Artificial diets containing naturally ¹³C-labelled sugar can isotopically mark the adults of some tephritids, but when sexually mature flies are released in the field, their food source usually changes from a C₄ to a C₃ plant. Consequently, the isotopic composition of flies can change toward the isotopic signatures associated with the new diet. For isotope labelling to be more meaningful in a pest management program that integrates the sterile insect technique, it is important to know the persistence of the carbon isotope marker in field-release sterile adults. Therefore, this study was intended to assess the degree of isotopic differentiation between wild samples of Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) and flies reared on different artificial diets, and to estimate the turnover of carbon in flies after shifts to different adult diets. The whole bodies of flies reflected the overall isotopic composition of their larval diets immediately after emergence. When the adult diet was switched, the δ¹³C signatures of flies changed rapidly for 6 to 8 d, then reached an isotopic equilibrium with the final diet. Depletions up to -5.6 (parts per thousand) were observed in the signatures of flies switched from a C₄ to a C₃-based diet. However, appropriate feeding on diets with C₄ sources ensured that the isotopic composition of larval diets was fixed in body structures of adult A. fraterculus, maintaining measurable ¹³C signals distinct from wild flies for 15 d after diet switching. No differences were found between males and females when they fed on different pre-release diets or after diet change. The ¹³C stable isotope proved to be a reliable marker for differentiating wild and laboratory-reared A. fraterculus flies.