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Highlights • Two commonly used methods for assessing the ecohydrological separation show inconsistent results. • A new method based on root zone isotope mass balance supports the ecohydrological separation at the studied humid site. • The ecohydrological connection occurs during snowmelt at the site. Abstract The ecohydrological separation between soil water sources for plant water uptake and groundwater recharge has been recently examined in various climate zones primarily based on isotopic composition of water. The existence of the ecohydrological separation has profound implications for mechanistic ecohydrological modeling and water resource management. However, it is still unclear when and where the ecohydrological separation occurs, especially in humid regions. In this study, high frequency sampling of precipitation, bulk soil water, groundwater and twig xylem water for hydrogen and oxygen isotope composition measurement was conducted in a humid subtropical site in the central southern China from March 2017 to April 2018. We examined evidence of the ecohydrological separation with three methods (dual-isotope space, line-conditioned excess (lc-excess), and the piecewise isotope balance (PIB) method). The results show that the isotopic composition of plant xylem and bulk soil water are not distinguishable from those of precipitation water on the dual-isotope space due to a weak evaporation effect at the study site, indicating that there is no evidence of the ecohydrological separation. However, the other two methods support the ecohydrological separation in this humid area, with the results from the PIB method revealing more temporal details. The present study suggests that the ecohydrological separation can happen in subtropical humid climate. It is more likely to occur in spring and winter at the study site when plant-accessible water pool has been replenished by antecedent precipitation, while ecohydrological connection seems to occur during winter snowmelt. With the limitations of three methods, the caution should be taken when only one method is applied in examining the ecohydrological separation in such an environment. [ABSTRACT FROM AUTHOR]