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We investigated how thermal stress may alter the locomotor phenotype of barramundi, Lates calcarifer , from genetically distinct northern (low latitude) and southern (high latitude) populations in tropical Australia. Following early growth and development under native (25 °C and 30 °C) and non-native (20 °C and 35 °C) temperatures, we observed distinct differences in the swimming performance of northern and southern individuals that were consistent with expectations based on local thermal adaptation. Southern population fish exhibited significantly faster swimming speeds (32.10 ± 0.33 cm·s–1, mass-adjusted mean ± 95% confidence limit) than their northern counterparts (28.58 ± 0.64 cm·s–1) under cold-stress (20 °C) conditions. Conversely, northern population fish performed significantly better (51.63 ± 2.1 cm·s–1) than their southern counterparts (44.18 ± 3.11 cm·s–1) under heat-stress (35 °C) conditions. We conclude that L. calcarifer display locomotor phenotypes thermally adapted to their local environment, with early growth under non-native temperatures leading to significantly reduced phenotypic fitness.