A comparative proteomic analysis of grain proteins during five grain developmental stages of wheat cultivar Chinese Spring (CS) and its 1Sl/1B substitution line CS-1Sl(1B) was carried out in the current study. A total of 78 differentially expressed protein (DEP) spots with at least 2-fold expression difference were detected by two-dimensional electrophoresis (2-DE). Among these, 73 protein spots representing 55 differentially expressed proteins (DEPs) were successfully identified by matrix-assisted laser desorption/ionization time-offlight tandem mass spectrometry (MALDI-TOF/TOF-MS). Differential protein spots between the two genotypes were analyzed by cluster software, which revealed significant proteome differences. There were 39 common spots (including 33 DEPs) that showed significant difference between the two lines across five grain developmental stages, of which 14 DEP spots (including 11 DEPs) were mainly involved in carbohydrate metabolism that were encoded by the genes on 1B chromosome while 25 DEP spots (including 12 DEPs) were mainly related to stress response and gluten quality that were encoded by 1S1 chromosome. These results indicated that the Sl genome harbors more stress and quality related genes that are potential valuable for improving wheat stress resistance and product quality.