A display substrate has a display area and a peripheral area. The display substrate includes a base, a first insulating layer disposed above the base, a first alignment pattern disposed in the peripheral area on a surface of the first insulating layer facing away from the base, and a second alignment pattern disposed in the peripheral area at a side of the first insulating layer away from the base. An orthographic projection of the second alignment pattern on the base and an orthographic projection of the first alignment pattern on the base have a non-overlapping region therebetween, and the second alignment pattern is in contact with the first insulating layer in the non-overlapping region. Adhesion between the second alignment pattern and the first insulating layer is greater than adhesion between the second alignment pattern and the first alignment pattern.