Abstract: Aims Recent advances in stable isotope techniques, in situ monitoring devices, and soil water extraction methods have increasingly supported the ecohydrological separation phenomenon: plants and streams appear to draw water from different soil reservoirs and return it to the hydrosphere. This review summarizes the evolution of the “two water worlds” (TWW) hypothesis since its initial proposal and discusses the latest research progress, particularly in small-scale field experiments, cross-scale analyses, and cross-ecosystem comparisons. Methods We systematically review the mechanisms and spatiotemporal dynamics underlying the separation of bound water and mobile water in the soil. We also discuss the connectivity between these water pools under various environmental conditions. Key issues, including identifying plant water source, standardizing soil water sampling methods, and addressing model uncertainty, are examined. Important findings Future research should focus on investigating plant water uptake mechanisms, improving water stable isotope monitoring techniques, integrating ecohydrological separation processes into hydrological models, and conducting cross-regional comparative studies. These perspectives provide valuable insights to guide further research in this field.

Key words: Ecohydrological separation, Two water worlds hypothesis, Bound water, Mobile water, Stable isotopes, Water source identification