Abstract. Increasing numbers of field studies have detected isotopic mismatches between plant trunk water and its potential sources. However, the cause of these isotopic offsets is not clear, and it is uncertain whether they occur during root water uptake or during water transmission from root to trunk. Thus, we measured the specific isotopic composition (δ2H and δ18O) of each component (e.g. bulk soil water, mobile water, groundwater, trunk water and root water of Salix matsudana Koidz trees) in the soil–root–trunk continuum with a resolution of about 3 days. We report three main findings. First, we detected a clear separation between the isotopic compositions of mobile water and bulk soil water, but the distinction between mobile water and bulk soil water gradually decreased with increasing soil depth. Second, root water composition deviated from bulk soil water isotopic composition but overlapped with the composition derived for less mobile water. The maximum differences in δ2H and δ18O between bulk soil water and root water were −8.6 ‰ and −1.8 ‰, respectively. Third, trunk water was only isotopically similar to root water at 100–160 cm depths, and it remained stable during the experimental period, suggesting that the trees consistently used the stable deep water source. In conclusion, the isotopic offset between bulk soil water and trunk water of S. matsudana reflected an isotopic mismatch between root water and bulk soil water associated with the heterogeneity of the soil water. Our results illuminate relationships between the isotopic compositions of soil waters of various mobilities, root water and trunk water that may be useful for advancing our understanding of root water uptake and transport.