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Stable isotopologues of water are widely used to derive relative root water uptake (RWU) profiles and average RWU depth in lignified plants. Uniform isotope composition of plant xylem water (δxyl) along the stem length of woody plants is a central assumption of the isotope tracing approach which has never been properly evaluated. Here we evaluate whether strong variation in δxyl within woody plants exists using empirical field observations from French Guiana, northwestern China, and Germany. In addition, supported by a mechanistic plant hydraulic model, we test hypotheses on how variation in δxyl can develop through the effects of diurnal variation in RWU, sap flux density, diffusion, and various other soil and plant parameters on the δxyl of woody plants. The hydrogen and oxygen isotope composition of plant xylem water shows strong temporal (i.e., sub-daily) and spatial (i.e., along the stem) variation ranging up to 25.2 ‰ and 6.8 ‰ for δ2H and δ18O , respectively, greatly exceeding the measurement error range in all evaluated datasets. Model explorations predict that significant δxyl variation could arise from diurnal RWU fluctuations and vertical soil water heterogeneity. Moreover, significant differences in δxyl emerge between individuals that differ only in sap flux densities or are monitored at different times or heights. This work shows a complex pattern of δxyl transport in the soil–root–xylem system which can be related to the dynamics of RWU by plants. These dynamics complicate the assessment of RWU when using stable water isotopologues but also open new opportunities to study drought responses to environmental drivers. We propose including the monitoring of sap flow and soil matric potential for more robust estimates of average RWU depth and expansion of attainable insights in plant drought strategies and responses. [ABSTRACT FROM AUTHOR]