• Stable isotope tracing of plant water use can illuminate plant water sources. • Xylem water isotope values showed strong sorting and niche segregation. • The majority of the observed species relied on 0.0–0.2 m depth soil water. • Tropical forest water uptake depth is largely driven by tree functional traits. Stable isotope tracing of plant water use can illuminate plant water sources. But to date, the number of species tested at any given site has been minimal. Here, we sample 46 tropical hardwood tree species in a 0.32 ha plot with uniform soils. Soil water was characterized at 6 depths at 0.2 m intervals down to 1 m and showed simple and predictable depth patterns of δ 2 H and δ 18 O, and simple and spatially uniform isotope composition at each depth. Nevertheless, tree xylem water δ 2 H and δ 18 O showed remarkable variation covering the full range of soil composition, suggesting strong sorting and niche segregation across the small plot. Wood density, tree size and mean basal area increment together explained approximately 55% of the variance of xylem water isotope composition through principal component analysis. A Bayesian mixing model was applied to the data and showed that sampled trees were either sourcing their water from very shallow or deep soil layers, with very little contribution from the middle portion of the soil profile. The majority of the observed species relied on 0.0–0.2 m depth soil water. This layer contributed approximately 75% of the xylem water which was significantly higher than the contributions from all other depths. The contribution from shallow soil was highest for trees with high wood density, slow-growing trees and small-sized trees. Our work suggests that stable isotope tracers may aid a better understanding of tropical forest water uptake depths and their relation to tree functional traits and potential hydrological niche segregation among co-occurring tropical species.