Research on the physiological thresholds of fish to hypoxia and temperature have focussed on short-term tolerances (<96 hours), with few researchers testing longer term exposures. Furthermore, fish exposed to persistent low levels of dissolved oxygen may be able to acclimate over time. Juvenile golden perch (Macquaria ambigua ambigua), silver perch (Bidyanus bidyanus) and Murray cod (Maccullochella peelii), key native freshwater species in the Murray Darling Basin, were acclimated for a longer time than generally used (30+ days) to different temperatures (20, 24 and 28°C) crossed with normal (6-8mg L-1) and hypoxic conditions (3-4mg L-1). Acclimation ability of Murray cod was also tested after 7 and 14 days of hypoxia exposure. Responses to hypoxia and thermal stress were measured for behaviour, mortality during the acclimation period and aerobic capacity (using resting respirometry). Murray cod and golden perch exhibited high tolerances to hypoxia during behavioural tests, while, silver perch did not survive more than 3 weeks exposure during the initial acclimation period. Longer term exposure to hypoxia improved the tolerances of golden perch during behavioural tests, however, Murray cod typically exhibited poorer tolerances the longer they were acclimated. Murray cod were most tolerant to hypoxia at the lowest temperature (20°C) and shortest exposure time. While prior exposure to hypoxia may allow fish to cope with hypoxic conditions better in the long-term, a short acclimation time resulted in the greatest tolerances for this study, suggesting resilience to hypoxia might decrease as a function of exposure time. Observed species-specific responses illustrate the need to consider tolerances and acclimation abilities of known sensitive species within a system to prevent mass mortalities of fish through better management of waterways.