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.