Studies of plant water sources generally assume that xylem water integrates the isotopic composition (δ 2 H and δ 18 O) of water sources and does not fractionate during uptake or transport along the transpiration pathway. However, woody xerophytes, halophytes, and trees in mesic environments can show isotopic fractionation from source waters. Isotopic fractionation and variation in isotope composition can affect the interpretation of tree water sources, but most studies to date have been greenhouse experiments. Here we present a field-based forensic analysis of xylem water isotope composition for 12 Eucalyptus tetrodonta and Corymbia nesophila trees . We used a 25-tonne excavator to access materials from the trees' maximum rooting depth of 3 m to their highest canopies at 38 m. Substantial within-tree variation occurred in δ 2 H (−91.1‰ to −35.7‰ E. tetrodonta ; −88.8‰ to −24.5‰ C. nesophila ) and δ 18 O (−12.3‰ to −5.0‰ E. tetrodonta ; −10.9‰ to −0.3‰ C. nesophila ), with different root-to-branch isotope patterns in each species. Soil water δ 2 H and δ 18 O dual isotope slopes (7.26 E. tetrodonta , 6.66 C. nesophila ) were closest to the Local Meteoric Water Line (8.4). The dual isotope slopes of the trees decreased progressively from roots (6.45 E. tetrodonta , 6.07 C. nesophila ), to stems (4.61 E. tetrodonta , 5.97 C. nesophila ) and branches (4.68 E. tetrodonta , 5.67 C. nesophila ), indicative of fractionation along the xylem stream. Roots of both species were more enriched in 2 H and 18 O than soil water at all sampled depths. Bayesian mixing model analysis showed that estimated proportions of water sourced from different depths reflected the contrasting root systems of these species. Our study adds evidence of isotopic fractionation from water uptake and along the transpiration stream in mature trees in monsoonal environments, affecting the interpretation of water sources. We discuss the findings with view of interpreting aboveground xylem water isotopic composition, incorporating knowledge of root systems. • Isotopic fractionation of xylem water may affect plant water source identification. • We analysed xylem δ 2 H and δ 18 O from roots to branches in mature trees in a savanna. • Fractionation increased from below- to aboveground xylem in the dual isotope space • Root structure assessment helped clarify aboveground interpretation of water use. • Future studies should consider xylem water fractionation and include plant traits.