Tree-based intercropping (TBI) systems induce microclimate modifications that can enhance crop performance through water use efficiency. However, the extent of this efficiency is dependent on water acquisition strategies of trees in such agroforestry systems. Charting water uptake zones at the tree-crop root interface in TBI systems is essential for managing water resources, particularly with increasing water stress from a changing climate. In this study, we identify the water uptake zones of Juglans nigra (walnut) and Populus sp. (hybrid of Populus deltoids X Populus nigra clone DN-177) (poplar) intercropped with winter barley (Hordeum vulgare) in a temperate TBI system. Water isotopic analysis using δ18O was conducted on paired soil water and tree xylem water samples in early season (pre-crop) and late season (with crop) collected from the Agroforestry Research Station in Guelph, Ontario, Canada. Two approaches for identifying the water uptake zones were used: direct inference and multiple source mass-balance analysis. Both approaches showed that poplar exhibits a dominant soil water uptake zone at 20 cm in the soil profile in the early season, while the dominant walnut uptake zone was higher in the soil profile at 10 cm. Depth of water uptake was lower in the soil profile (40–70 cm) in the late season for both species. This study indicates: (i) tree species dependent water acquisition zones, and (ii) a shift in tree water acquisition to deeper in the soil profile over the growing season. Using isotopic techniques, we suggest that poplar and walnut root activity stratify below the crop root zone later in the growing season, potentially reducing competition for water resources.