A display substrate includes a substrate on which a first pixel area including a first light-blocking region, and a second pixel area adjacent to the first pixel area and including a second light-blocking region are defined, an insulating layer in the first and second light-blocking regions, a black matrix pattern layer on the insulating layer, a first column spacer in the first light-blocking region and protruding from the black matrix pattern layer, and a second column spacer in the second light-blocking region and protruding from the black matrix pattern layer. A height of a top surface of the first column spacer is different from a height of a top surface of the second column spacer, where the heights are taken with respect to the substrate.