Abstract: Aiming at the problem of surface subsidence after underground coal mining, to accurately locate the key stratum for subsidence reduction by grouting in overlying rock separation zones and effectively control surface subsidence and its secondary adverse impacts, a comprehensive identification system for key strata in overlying rock separation zones integrating the elastic foundation beam theory, 3D seismic data inversion and finite difference numerical simulation was proposed. Based on the elastic foundation beam theory, a mechanical model of deflection of zonal elastic foundation beams was established, and the discriminant formula for key strata and the stability evaluation model for grout isolation layers were derived. Using the results of stratum lithology inversion from 3D seismic data and combining numerical simulation to analyze the evolutionary characteristics of overlying rock separation, the spatiotemporal evolution laws of the overlying rock pressure relief zone, vertical displacement field and plastic zone were revealed to determine the grouting horizon for separation zones. Taking the 11090 working face in the Xin’an Coalfield as an engineering case, the theoretical calculation value of the development height of the water flowing fractured zone was 78.60 m, and the numerical simulation result was 80–100 m; numerical simulation revealed that the upper boundary of the overlying rock pressure relief zone was located in the rock stratum with a buried depth of 480–500 m. By comprehensively using the discriminant formula for key strata, the theoretical value of the height of the water flowing fractured zone, numerical simulation results and the safety factor evaluation model for the stability of grout isolation layers, the ultra-thick mudstone stratum at 147.26 m above the coal seam, with a buried depth of 482.94 m and a thickness of 63.62 m, was determined as the target grouting layer. For this target layer, 8 surface boreholes with the final hole at 1 m from the bottom of the stratum were designed, forming a grouting technology of paired arrangement and phased grouting; meanwhile, 83 measuring points were arranged to carry out 17 phases of surface movement monitoring. The monitoring results showed that the maximum subsidence value was 231.2 mm, the maximum inclination was 0.6 mm/m, and the maximum deformation value was 0.3 mm/m. Compared with the theoretical maximum subsidence value of 879.3 mm in non-grouting mining, the subsidence reduction rate reached 73.1%, and the building damage grade was reduced from Grade II to Grade I. The study confirms that the comprehensive identification method integrating 3D seismic technology and numerical simulation based on the elastic foundation beam theory can accurately locate the target grouting layer for overlying rock separation zones; the optimized grouting technology system adapted to the ultra-thick mudstone key stratum can realize the efficient control of surface subsidence.