The illegal pet trade facilitates the global dispersal of invasive alien species (IAS), providing opportunities for new pests to establish in novel recipient environments. Despite the increasing threat of IAS to the environment and economy, biosecurity efforts often lack suitable, scientifically-based methods to make effective management decisions, such as identifying an established IAS population from a single incursion event. We present a proof-of-concept for a new application of a stable isotope technique to identify wild and captive histories of an invasive pet species. Twelve red-eared slider turtles (Trachemys scripta elegans) from historic Australian incursions with putative wild, captive and unknown origins were analysed to: (1) present best-practice methods for stable isotope sampling of T. s. elegans incursions; (2) effectively discriminate between wild and captive groups using stable isotope ratios; and (3) present a framework to expand the methodology for use on other IAS species. A sampling method was developed to obtain carbon (δ13C) and nitrogen (δ15N) stable isotope ratios from the keratin layer of the carapace (shells), which are predominantly influenced by dietary material and trophic level respectively. Both δ13C and δ15N exhibited the potential to distinguish between the wild and captive origins of the samples. Power simulations demonstrated that isotope ratios were consistent across the carapace and a minimum of eight individuals were required to effectively discriminate wild and captive groups, reducing overall sampling costs. Statistical classification effectively separated captive and wild groups by δ15N (captive: δ15N‰ ≥ 9.7‰, minimum of 96% accuracy). This study outlines a practical and accessible method for detecting IAS incursions, to potentially provide biosecurity staff and decision-makers with the tools to quickly identify and manage future IAS incursions.