The division scheme discussed in this study is based on the characteristics and spatial distribution of aquatic ecosystems; the scheme takes into account natural geographic features, water resources, hydrological characteristics, ecosystem functions, environmental and socio-economic conditions, and the impact of water conservancy projects on watersheds. It is based on divisions within China based on natural geography, ecological function, water resources, and water functions. This study proposes the establishment of national water eco-region divisions, including an indexing system, specific methods for establishment of divisions, and a sequence of steps for dividing water eco-regions. This study also divided watersheds nationwide into various water eco-regions, which were subsequently evaluated and verified using economic test procedures. The proposed water eco-regions would provide a viable basis for the comprehensive utilization of water resources, ecological protection, and economic and social development, for general planning of nationwide watersheds. There are three landforms extending from west to east in China, together with a dry and wet area stretching from the northwest to the southeast. Water eco-regions take into account existing physical geography, climate, water resources, and ecological conditions, as well as national ecological function zoning and water districts. From the perspective of systems analysis, we propose an indicator system that includes three layers, namely target, feature, and index layers; these provide a reflection of the spatial distribution of regional natural geographical and climatic conditions, water resources, aquatic ecosystems, human activities, the layout of economic and social structures, and ecological differences. The index layer included 11 indices; the key indicators among these for division of water eco-regions are landform, vegetation cover, and depth of annual runoff. A set of secondary indicators was used to adjust and correct the preliminary subdivisions; this included altitude, the status of aquatic systems, important protected species, the distribution of rivers and lakes, the location of reservoirs, administrative divisions, population density, and the distribution of large cities. The Gini coefficient (also known as the Lorenz factor) for economic income distribution was applied to evaluate the rationality of the proposed aquatic ecosystems division scheme. The eco-region scheme was adjusted and corrected according to evaluation results. The method took into account total water resources of each eco-region as an evaluation index, as well as rainfall, population density, and the NDVI value of each eco-region as distribution indices; these were used to verify the division schemes using the Gini coefficient and Lorenz curves. Results indicated that the division scheme was reasonable when the Gini coefficient for water resources and amount of rainfall was 0.10, when the coefficient for water resources and population density was 0.40, and when that for water resources and NDVI was 0.32. We propose a two-grade eco-region division system for national aquatic ecosystems. This includes four first-grade aquatic eco-regions, namely the east eco-region, middle eco-region, northwest eco-region, and southwest eco-region. These serve macro-economic management purposes, as well as fulfilling overall layout needs for the development and utilization of water resources and aquatic ecosystems protection. Based on water resource management and facilities for ecological protection and restoration in watersheds, 34 second-grade aquatic eco-regions are proposed, including 13 in the east eco-region, 10 in the middle eco-region, 6 in the northwest eco-region, and 5 in the southeast eco-region. The proposed national water eco-division scheme provides a scientific basis for national ecological protection and construction planning, for maintaining regional ecological security and promoting sustainable social and economic development, and for providing management information and tools for environmental management and decision-making departments.