AbstractDue to global biodiversity declines, conservation programmes have increasingly had to consider reintroducing captive animals into the wild. However, reintroductions often fail as captive individuals may be naïve to predators and do not recognise or respond appropriately to predatory cues, contributing to high mortality rates soon after release. This study evaluates differences in predator‐response behaviours between individuals from three experimental groups, a captive population (n = 13), a semi‐wild population (i.e. raised in captivity and successfully released; n = 6) and a wild population (n = 2) of cheetah (Acinonyx jubatus) to an artificially simulated auditory threat of lions (Panthera leo), a larger, natural predator in South Africa. Such comparisons improve our understanding of differences between captive and wild behaviours and provide an aspect to evaluating the relative success of reintroduction programmes. Changes in the proximal distance, the latency of approach and hesitation towards both control (African bush cricket, Acanthoplus discoidalis) and treatment (lion) auditory cues were observed for 29 cheetah from captive, semi‐wild and wild populations in at least three trial replicates each. Overall, captive individuals consistently displayed poor predatory‐response behaviours, approaching the treatment as often as the control, as well as spending more time near the stimulus (<10 m) and hesitating more often than semi‐wild cheetah, which could distinguish between the control and treatment, consistently fleeing from the latter with little hesitation. Repeatability analyses indicated that these behavioural responses to predatory cues could not be explained by individual personality and between‐trial learning comparisons showed no evidence of habituation. Our findings demonstrate how a priori testing for predator naïvety could inform future introductory decisions and thereby increase post‐release survival rates, significantly improving the efficacy of reintroduction strategies. We, therefore, emphasise the importance of such research and screening in highly threatened species, such as cheetah, where reintroduction from captivity has become a necessary consideration.