The regionalization methodology systems of aquatic ecosystems consist of guiding concepts, fundamental principles, regionalization approaches, nomenclature and code rules. The core of the guiding concepts is the scientific outlook on development and idea of harmonious coexistence between human beings and nature; the fundamental principles include region relativity principle, coordination principle, dominant function principle and graded regionalization principle; the regionalization approaches, nomenclature and code rules vary with the grade and scale of the study areas. In this paper, according to the thought of two-level regionalization, the scheme of regionalization of aquatic ecosystems which contains 6 regions and 34 subregions in China is presented. Based on the classification of aquatic ecosystem service functions, i.e., aquatic ecosystems conservation (water resource protection, soil and water conservation and species conservation), aquatic ecosystems regulation (flood regulation and habitat maintenance), and aquatic ecosystems service (product supply and landscape protection), the three-level regionalization approach is applied in the Huaihe River Basin. In this scheme, the whole basin covers 2 regions, 3 subregions and 21 sections in all.