The present invention provides a display substrate and a manufacturing method thereof, and a driving method of a display substrate. In the display substrate, a plurality of pixel groups are repeatedly arranged on a substrate base, each of the pixel groups comprises four first subpixels, four second subpixels, four third subpixels and four fourth subpixels, the subpixels in each of the pixel groups are arranged in a 4×4 matrix, each row of subpixels in each of the pixel groups include one first subpixel, one second subpixel, one third subpixel and one fourth subpixel, the first subpixel, the second subpixel, the third subpixel and the fourth subpixel in different rows of subpixels in each of the pixel groups are arranged successively in different orders, and subpixels with the same color are not located in the same column so that the subpixels are distributed uniformly.