The hypothesis of ecohydrological separation (ES) proposes that the water contained in surface soils is not uniformly extracted by root water uptake nor uniformly displaced by infiltration. Rather vegetation selectively removes water held under tension, and water infiltrating wet soil will bypass much of the water‐filled pore space. Methodological differences across previous studies have contributed to disagreement concerning the prevalence of ES. We measured stable isotopes of O and H in precipitation, snowpack, canopy throughfall, and stream water over a period of 18 months in a temperate catchment. At six locations across a wetness gradient, we sampled bulk soil water isotopes weekly and xylem water of Eastern hemlock and American beech stems seasonally. We used these observations in a soil column model including StorAge Selection functions to estimate the isotopic composition and ages of groundwater recharge and ET. Our findings suggest ES may exist with spatial and temporal heterogeneity. Root water uptake ages possibly vary between Eastern hemlock and American beech, suggesting functional strategies for water uptake may control the presence of ES. Newly infiltrated water bypassing the shallow soil was the most likely explanation for bulk soil isotopic measurements made at upslope locations during the winter and summer seasons, whereas rapid displacement of stored soil water by infiltrated waters was the most likely during the spring and fall seasons. Future research incorporating high temporal frequency soil and plant xylem water isotopic measurements applied to StorAge Selection functions may provide a useful framework for understanding rooting zone isotope dynamics.

The Hammond Hill Research Catchment (HH) is a small (120 ha), temperate, second order tributary to Six Mile Creek, Cayuga Lake, and the Great Lakes (42.42°, −76.32°). The HH has been monitored since January 2017 for the purpose of understanding how recent infiltration mixes with antecedent soil water on hillslope forest floors and the spatial and temporal patterns of Root Water Uptake (RWU) by temperate northeastern US tree species (eastern hemlock [Tsuga canadensis], American beech [Fagus grandifolia], and sugar maple [Acer saccharum]). These data are informing us about the hydrologic consequences of anticipated tree species composition change and supporting the development of more refined ecohydrological models. The glaciated catchment is underlain by a shallow confining siltstone layer (1–1.5 m depth) and densely covered with an approximately 60 year old regrowth mixed species forest of hemlock, beech, and other deciduous tree species common to the northeastern US. Current datasets from the HH include precipitation snow water equivalent, discharge, and associated isotopic water compositions, δ2H & δ18O. Measurements of (top 10 cm) soil water content, as well as bulk soil water and hemlock and beech xylem isotopic compositions are made at several locations across a topographic wetness gradient. The near‐term role of the HH is to support an understanding of the environmental and ecological drivers of plant RWU competition. All data from the HH are publicly available.