Abstract: Water inrush from the separation layers of overlying strata, as a common but severe hydraulic disaster, occurs frequently in the eastern and western mines of China in recent years. The evolution of mining overburden structure is of great significance to reveal the mechanism of water inrush from separation layers and to predict and prevent the disaster. To reveal the evolution mechanism of water inrush from separation layers in different engineering geological conditions, the composite strata related to water inrush from separation layers, involving the upper strata, water-filled separation layers and the lower water-resisting layers, are collectively referred to as “Water-inrushing Separation Zone, WSZ for abbreviation”. The structural mechanical model of the WSZ causing water inrush was established. According to the different factors affected by the breaking laws of upper strata and lower water-resisting layers, the formation mechanism and mechanical conditions of five types of roof water inrush, including hydrostatic water inrush, dynamic water inrush, water inrush induced by multiple coal seam superimposed mining, water flowing through water-conducting fractures and no water inrush from separated layer, were analyzed. Furthermore, the temporal and spatial evolution of the water-conducting fractured zone and the water inrush from the separation layers during mining was studied. Based on the mining conditions of working face, overburden conditions, the position of the WSZ, and microseismic monitoring, the formation conditions of water inrush from the separation layers were analyzed, and the evolution mechanism was revealed. Additionally, the working faces of the Cuimu colliery and the Zhaoxian colliery in Shaanxi Province were analyzed as case studies, and the prediction method of water inrush from the separation layers of working faces was proposed. The results show that the five types of water inrush are essentially related to the breaking law of the WSZ under the action of different water pressures and the supporting forces from the underlying strata with the face advance. The statistical data indicates that only with a certain relation between the mining height(M) and the distance of the WSZ to coal seam(H), the basic conditions of the WSZ can be satisfied. On the basis of the mechanical criterion of the WSZ, the water inrush from the separation layers in the working face 21306 of the Cuimu colliery is caused by the fractured upper strata. During the water inrush, the frequency of microseismic events whose energy is more than 10⁴ J increased, and the total energy and average energy per day increased significantly. Whereas, the water inrush of the working face 1304 of the Zhaoxian colliery was caused by the fractured lower water-resisting layer. During the water inrush, the frequency of microseismic events whose energy is less than 10³ J increased, and the corresponding total number of microseismic events increased significantly while the average energy per day decreased.