The Yangtze River Basin has long been a region with striking conflicts between the natural resource needs of economic growth and those of biodiversity conservation. Large-scale infrastructure development and over-exploitation of natural resources have resulted in ecosystem degradation that increasingly threatens the growing number of endangered species in the Basin. To conserve biodiversity in this Basin, priority conservation areas (PCAs) were identified based on an integrated method of systematic conservation planning with expert participation. According to the distribution of 1015 key protected species, a total of 28 PCAs were proposed, including 13 for primarily terrestrial ecosystems and 15 for aquatic ecosystems. Aiming at the existing threats to biodiversity, it is recommended that systematic surveys be conducted to assess biodiversity status and its threats, and systematic planning of nature reserves be carried out mainly based on each PCA, the negative impacts on biodiversity resulting from construction and resource development be mitigated by controlling water pollution, guaranteeing supply of water for maintenance of ecosystem, and strengthening of management of tourism and mining activities within PCAs. These results will provide input for the WWF Yangtze River Eco-region Action Plan (2010-2015), and serve as a basis for government in making decision in the Basin.