The invention discloses a display substrate and a driving method thereof, and a display device. The display substrate includes a plurality of rows or columns of pixel units, wherein each row or column of pixel units include first pixel units and second pixel units which are arranged alternately, one first pixel unit and one second pixel unit are composed of three sub-pixels, and wherein the first pixel unit includes a first sub-pixel and a second sub-pixel, the second pixel unit includes the second sub-pixel and a third sub-pixel, and the first, second and third sub-pixels are arranged in turn. In the invention, three sub-pixels form two pixel units, thus the number of the sub-pixels needed to form a certain number of pixel units is decreased, and the manufacturing process is simplified and the defective rate of product is decreased.