Current land surface models assume that groundwater, streamflow and plant transpiration are all sourced and mediated by the same well mixed water reservoir-the soil. However, recent work in Oregon(1) and Mexico(2) has shown evidence of ecohydrological separation, whereby different subsurface compartmentalized pools of water supply either plant transpiration fluxes or the combined fluxes of groundwater and streamflow. These findings have not yet been widely tested. Here we use hydrogen and oxygen isotopic data (H-2/H-1 (delta H-2) and O-18/O-16 (delta O-18)) from 47 globally distributed sites to show that ecohydrological separation is widespread across different biomes. Precipitation, stream water and groundwater from each site plot approximately along the delta H-2/delta O-18 slope of local precipitation inputs. But soil and plant xylem waters extracted from the 47 sites all plot below the local stream water and groundwater on the meteoric water line, suggesting that plants use soil water that does not itself contribute to groundwater recharge or streamflow. Our results further show that, at 80% of the sites, the precipitation that supplies groundwater recharge and streamflow is different from the water that supplies parts of soil water recharge and plant transpiration. The ubiquity of subsurface water compartmentalization found here, and the segregation of storm types relative to hydrological and ecological fluxes, may be used to improve numerical simulations of runoff generation, stream water transit time and evaporation-transpiration partitioning. Future land surface model parameterizations should be closely examined for how vegetation, groundwater recharge and streamflow are assumed to be coupled.