Naturally occurring elemental signatures (or composition) of otoliths may enable aquaculture and wild-caught yellowtail kingfish (Seriola lalandi) to be distinguished from one another, so that in the event of aquaculture escapes, escaped fish could be identified. Yellowtail kingfish were obtained from aquaculture ventures in three regions of Spencer Gulf and from nine areas external to aquaculture throughout the inner and outer Spencer Gulf, South Australia. Elemental signatures (Mg, Mn, Sr, Ba) of otoliths were analysed via LA-ICP-MS (laser ablation-inductively coupled plasma-mass spectrometry). A highly significant difference was detected between elemental signatures of the aquaculture and wild-caught fish, but this difference was largely attributable to wild fish from Port Augusta being different from the other groupings. For individual elements, it was difficult to detect differences between aquaculture and wild-caught fish, as there was considerable variation between locations within each group. When comparisons between aquaculture and wild fish were made of the inner and outer Spencer Gulf, aquaculture fish could be distinguished from wild fish and classified correctly with a high degree of accuracy (82–100%); the same level of accuracy was not achieved with wild fish. Comparison of elemental transects showed considerable variability between locations, with no features distinguishing aquaculture or wild-caught fish. It would be beneficial to combine naturally occurring signatures with other identification approaches (e.g. artificial elemental signatures) to accurately distinguish aquaculture from wild-caught fish. The impacts of escaped fish could then be determined.