The present disclosure provides a display substrate, a display device and a method for recognizing a positioning mark. The display substrate includes a substrate, and a first pattern and a second pattern formed on a first side of the substrate; the second pattern is disposed between the first pattern and the substrate, and the first pattern has a first through hole directly facing the second pattern; the first pattern and the second pattern are formed of materials having different reflectances, and the first through hole serves as a positioning mark on a first side of the substrate for positioning the first side of the substrate.