Eucalyptus plantations are expanding in response to global wood demand but are raising concerns about their impacts on water supplies. Sustainable plantation management in areas with water conflicts will require accurate assessments of tree and stand water use. Thermal dissipation probes have been used to estimate tree water use, but recent work suggests that species-specific calibrations may be required to obtain accurate results. In this study, we quantified sap flux density (SFD) in 2-year-old Eucalyptus globulus Labill (Eg) and E. nitens x globulus (Eng) species using the thermal dissipation method developed by Granier. For each species we compared the original Granier equation with species-specific calibrations using whole tree potometers over a 36-h period. Our results showed that on average, Granier's original equation significantly underestimated SFD in both species, and when scaled to the stand level, tree transpiration (Ec) was significantly lower compared to onsite calibrations. The Granier method also underestimated nocturnal transpiration for both genotypes. Measured calibration coefficients were similar and not statistically different between Eg and Eng. These results highlight the importance of species-specific calibrations using thermal dissipation probes for Eucalyptus species to improve stand water use estimates and inferences about ecological impacts.