Insect water balance plays an important role in determining energy budgets, activity patterns, survival, and population dynamics and, hence, geographic distribution. Tsetse (Glossina spp.) are important vectors of human and animal disease occupying a wide range of habitats in Africa and are notable for their desiccation resistance in xeric environments. Here, we measure water balance and related traits [water loss rate (WLR), body water content (BWC), body lipid content (BLC) and body mass] in adult flies across a range of temperature (20-30 degrees C) and relative humidity (0-99%) combinations in four tsetse species from both xeric and mesic habitats. WLRs were significantly affected by measurement under different temperature and relative humidity combinations, while BWC, BLC, and body mass were less affected. These results provide support for mass-independent inter- and intra-specific variation in WLRs and survival times. Furthermore, water balance responses to variation in temperature and relative humidity are complex in Glossina, and this response varies within and among species, subgroups, and ecotypes in terms of both magnitude of effects and the direction of change. Different effects of temperature and relative humidity within and among experimental conditions and species suggests cuticular permeability and saturation deficit are likely to be key factors in forecasting tsetse water balance responses to climate variability. This complicates potential forecasting of tsetse distribution in the face of climate change.