In a method of manufacturing a display substrate and a method of manufacturing a display panel, the display substrate includes a color filter layer disposed on a base substrate within a pixel area, a first organic insulating pattern disposed on a first boundary area between adjacent pixel areas, a pixel electrode disposed on the color filter layer, and a first blocking pattern disposed on the first organic insulating pattern. Accordingly, an organic insulating layer corresponding to the pixel area is removed so that deterioration of the display quality by impurities generated from the organic insulating layer may be minimized. In addition, a stepped portion of a blocking pattern disposed between a pixel area and a boundary area of a plurality of the pixel areas is reduced so that motion blurring of a liquid crystal may be prevented.