a b s t r a c tBecause of limitation of manpower, funding, and land available in conservation, the problem of how to select essential regions to establish protection systems for biodiversity maintenance has been widely discussed. In an effort to address the problem, this study has aimed to select a set of priority areas and to determine their priority order by quantifying human disturbances for each area in the Yangtze River Basin (YRB). This basin covers 2.143 million km 2 , or more than 20% of China's territory. The habitats of 627 indicator species were predicted as a proxy for biodiversity. A conservation planning tool, MARXAN, was used to determine the optimal set of planning units, and three different target scenarios were generated. In addition, under the assumption that if two areas have equal value for conservation, the one suffering more severe disturbance needs more urgent protection than the other, priority ranking analysis was carried out using a 6-12-1 BP artificial neural network. Then hierarchical cluster analysis was applied to the classifications of human disturbances to formulate more detailed conservation strategies. By integrating the degree of irreplaceability of each unit, expert experience, and mountain boundaries, 17 biodiversity priority areas containing 33,200 units over an area of 0.83 million km 2 were defined. These areas also protected 56% of 32 types of rare forest ecosystem and 76.4% of six types of rare grassland ecosystem on average. According to the evaluation of human impact, a priority order and five types of human disturbance areas were generated. Some protection gaps were also identified, such as the northern part of the Wuyi Mountains. Moreover, the determination of priority nature conservation areas on a large scale can be used to influence the building of a well-connected protection network in each individual area, so that effective genetic communication can occur between species or groups of species. Conservation decisions focusing on the dominant impact factors that are threatening biodiversity sustainability are required as well.