Separation and analysis of water-soluble proteins (WSP) are important in understanding wheat grain proteome fundamentals. However, due to their high degree of heterogeneity and complexity in the compositions, separating WSP is generally difficult and relevant methodologies are not efficiently developed yet. Capillary electrophoresis (CE) is one of the analytical methods currently used for protein separation and characterization. In the present study, a CE method is established for rapidly separating and characterizing WSP of wheat grains. The established method was tested in various applications including wheat variety and germplasm identification as well as protein synthesis and accumulation studies during different grain development stages subject to genotypic and environmental variations. As results, the characteristic CE patterns of a range of bread wheat cultivars and related species were readily identified. The synthesis and accumulation patterns of wheat WSP during developing grains as well as their stabilities in different environments were also investigated. The technical advancements present in this article appear to be useful for wheat cultivar and germplasm identification as well as genetics and breeding research.