Water resources management of the Tarim River Basin in China is hampered because it is poorly gauged, leading to inadequate understanding of the components of the hydrological cycle. This is an important problem to both ecology and society in this arid basin. In this paper, we attempt to investigate the isotopic characteristics of the surface water and groundwater in the Tarim River Basin and estimate the water evaporation loss from lakes. By measuring the water stable isotope composition and by using a mass balance model, we analyzed the spatial characteristics of the isotope composition, and isotopic water lines for surface water and groundwater were determined. Phreatic water evaporation was verified on the basis of the isotope composition results. The spatial variability of the Aksu River exhibited a ‘toward lighter–heavier’ change along the main stream for both δ18O and δD, and the distributions could be described by a cubic equation. The isotope composition of the evaporation vapour (δE) of the Aksu River decreased with distance. No regular trend was observed for the Tarim River between the isotope composition and distance, whereas δE appeared to exhibit the same characteristics. This study is the first to use the isotopic method in the evaporation research for Bosten Lake. The water fraction lost [evaporation (E)/inflow (I)] in October was 0·25, which suggests that 0·68 × 108 m3 input water was lost to evaporation. Copyright © 2013 John Wiley & Sons, Ltd.