The disclosure provides a display substrate motherboard, a manufacturing method thereof, a display panel motherboard and a manufacturing method of a display substrate. The display substrate motherboard includes a base substrate including multiple substrate areas, each substrate area including a display region and a pad region; a display structure layer located on the base substrate and including a pixel defining layer and multiple film layers located between the pixel defining layer and the base substrate, a blocking region is arranged between any two adjacent substrate areas; a portion of the display structure layer in the blocking region has a first groove therein, a portion of the display structure layer between the first groove and the pad region adjacent thereto constitutes a first spacer region; a thickness of the portion of the display structure layer in the first spacer region is less than that in the display region.