An organic light emitting display substrate comprises a substrate, a first pixel defining layer provided on the substrate and a second pixel defining layer provided on the first pixel defining layer. The first pixel defining layer includes a plurality of first openings, and the second pixel defining layer includes a plurality of second openings in one-to-one correspondence with the plurality of first openings. An edge of each of the plurality of second openings extends outwards beyond an edge of the corresponding first opening so as to expose a portion of the first pixel defining layer, and a difference in length between each of the plurality of second openings and the corresponding first opening is greater than a difference in width between the second opening and the corresponding first opening.