Based on the researches on biodiversity characteristics in Northeast China, The value of irreplaceability for the conservation planning units are calculated by using systematic conservation planning and software C-plan. Four hotspot areas are identified, including northwestern of Changbai Mountain, northern Daxinganling Mountain, the transitional zone between western grassland and Daxinganling forest mountain area, and the wetlands around the center of Songnen plain. The most priority area for conservation is 194,900 km2, 20.91% of the whole research area. In the meantime, a gap analysis for biodiversity conservation has been conducted in this region based on the results of hotspots′ assessment, The results show that there are three evident gaps for protection based on an analysis of currently existing national nature reserve, including Changbai Mountain area, northern of Daxinganling Mountain area, the transitional area between western grassland and Daxinganling forest mountain area. Suggestions for establishing new PAs and eco-corridors have been put forward in this paper.