A display substrate, a display device and a driving method thereof are provided. The display substrate includes a plurality of gate lines and a plurality of data lines. The plurality of gate lines crosses the plurality of data lines to define a plurality of sub-pixel regions. Each of the plurality of sub-pixel regions includes a TFT connected to one of the plurality of data lines. Two rows of sub-pixel regions are arranged between every two adjacent gate lines, and two data lines of the plurality of data lines are arranged between every two adjacent columns of sub-pixel regions. TFTs in two adjacent rows of sub-pixel regions at two sides of one gate line of the plurality of gate lines are connected to the gate line and connected to different data lines.