Tropical coastal systems are particularly prone to periods of environmental hypoxia, which can result from organismal respiration as well as thermal stratification, and may be further exacerbated by anthropogenic disturbances. In this study, we used five genetically distinct sub-populations of Australian barramundi (Lates calcarifer) to examine the extent of intraspecific variability in hypoxia tolerance. Fish were maintained at two temperatures (26 or 36°C), representing the seasonal thermal range for this species across its tropical distribution in Australia. All fish maintained a constant oxygen consumption rate as air saturation of the water decreased from 100% down to a critical oxygen saturation ([O2]crit) of 15.44 ± 3.20 and 21.07 ± 3.92% (means ± SD) at 26 and 36°C, respectively. Mean [O2]crit, used as a performance measure of hypoxia tolerance, did not differ between sub-populations. No differences were found for resting between sub-populations at 26°C, but modest differences were detected between two sub-populations at 36°C (3.36 ± 0.62 and 2.83 ± 0.27 mg O2 kg−1 min−1 for Gladstone and Broome sub-populations, respectively). Resting was lower for sub-populations at 26°C (1.46 ± 0.26 mg O2 kg−1 min−1) than at 36°C (3.10 ± 0.43 mg O2 kg−1 min−1), with a temperature coefficient (Q10) of 2.12 ± 0.30. We conclude that both hypoxia tolerance and resting are conserved across the distribution of barramundi in Australia, which reflects the capacity of this species to cope in environments with large fluctuations in both temperature and dissolved oxygen.