Comparative proteomics of seed filling between yellow-seeded progeny from somatic hybrids Brassica napus-Sinapis alba and black-seeded parent (B. napus) were taken out using two-dimensional electrophoresis (2-DE). The process indicated distinct differences in 2, 3, 4, 5, 6 weeks after fertilization (WAF) and mature seed. A total of 8 out of the 27 discriminate proteins were identified by mass spectrum analysis and MASCOT comparison, including protein kinase, enolase, triosephosphate isomerase, and dioxygenase. PCR primers contrived for the putative genes were applied for further identification of progenies and both parents, which indicated that spot A3-5 might be the novel protein of intergeneric hybrid, i.e., A5-2 derived from S. alba. Applying these specific primers, this study demonstrates that the new yellow-seeded germplasm is different from the existing yellow seed materials of rapeseed.