Water transpired by trees has long been assumed to be sourced from the same subsurface water stocks that contribute to groundwater recharge and streamflow. However, recent investigations using dual water stable isotopes have shown an apparent ecohydrological separation between tree-transpired water and stream water. Here we present evidence for such ecohydrological separation in two tropical environments in Puerto Rico where precipitation seasonality is relatively low and where precipitation is positively correlated with primary productivity. We determined the stable isotope signature of xylem water of 30 mahogany (Swietenia spp.) trees sampled during two periods with contrasting moisture status. Our results suggest that the separation between transpiration water and groundwater recharge/streamflow water might be related less to the temporal phasing of hydrologic inputs and primary productivity, and more to the fundamental processes that drive evaporative isotopic enrichment of residual soil water within the soil matrix. The lack of an evaporative signature of both groundwater and streams in the study area suggests that these water balance components have a water source that is transported quickly to deeper subsurface storage compared to waters that trees use. A Bayesian mixing model used to partition source water proportions of xylem water showed that groundwater contribution was greater for valley-bottom, riparian trees than for ridge-top trees. Groundwater contribution was also greater at the xeric site than at the mesic–hydric site. These model results (1) underline the utility of a simple linear mixing model, implemented in a Bayesian inference framework, in quantifying source water contributions at sites with contrasting physiographic characteristics, and (2) highlight the informed judgement that should be made in interpreting mixing model results, of import particularly in surveying groundwater use patterns by vegetation from regional to global scales.