The ecohydrological-separation (ES) hypothesis is that the water used for plant transpiration and the water used for streams and groundwater recharge comes from distinct subsurface compartmentalized pools. The ES hypothesis was first proposed in a study conducted in the Mediterranean climate region, based on the stable isotope method in 2010. To date, the ES hypothesis has proven to be widespread around the world. The ES hypothesis is a new understanding of the soil water movement process, which is different from the assumption that only one soil reservoir in the traditional hydrology. It is helpful to clear the water sources of plants and establish a new model of the ecohydrological process. However, the theoretical basis and mechanism of the ES hypothesis are still unclear. Therefore, we analyzed the characteristics of ES phenomenon in different climatic regions, summarized the research methods used for the ES hypothesis, concluded the definitions of tightly bound water and mobile water, discussed the mechanism of isotopic differences of different reservoirs and their impacts on ES evaluation and pointed out the existing problems of the ES hypothesis. Future research should focus on the following three aspects: (a) detailed analysis of ES phenomenon characteristics of different plant species in different climatic regions; (b) further understanding of the ES phenomenon mechanism; (c) improvement of the experimental methods.