Atlantic tarpon Megalops atlanticus are highly migratory sportfish that support recreational fisheries throughout their range. In US waters, juveniles can be found in coastal and estuarine habitats along the Gulf of Mexico and Atlantic seaboard, with temperature limiting their northern latitudinal distribution. Juveniles may overwinter in these areas during the first several years of life. Low temperatures are known to cause mortality in adults, but the challenges of temperature are less understood for juveniles. Furthermore, salinity, which can change dramatically in these habitats, may have a synergistic effect with temperature. To examine the physiological effects of temperature and salinity on juvenile tarpon, wild fish were acclimated to a range of conditions that potentially occur in the northern range of their estuarine habitats. The haematology of juvenile tarpon was examined in two salinity (≤2 and ≥30 ppt) and temperature (15 and 25°C) treatments, followed by a low‐temperature tolerance test. After 2 weeks in treatment conditions, blood samples were analysed for haematocrit, pH, red blood cell concentration, haemoglobin content and plasma osmolality. Increased plasma osmolality was observed in fish at low temperature (15°C compared to 25°C) and at high salinity (≥30 ppt compared to ≤2 ppt). Blood pH was increased at 15°C compared to 25°C, with the highest pH at 15°C and low salinity. Haemoglobin, haematocrit and red blood cell concentration were higher at 25°C than 15°C, with haemoglobin lowest at 15°C and low salinity. For the low‐temperature tolerance test, all fish were acclimated to 15°C for 2 weeks, then transferred to separate tanks where temperature was gradually decreased at 0.9 ± 0.1°C/h until fish lost equilibrium. Fish at low salinity lost equilibrium more rapidly (1 ppt, 12.65 ± 0.46°C) than fish at high salinity (30 ppt, 11.26 ± 0.14°C). The results indicate juvenile tarpon are susceptible to low temperature, which is exacerbated by low salinity, findings useful in the assessment of juvenile tarpon overwintering habitat.