Proteomic technologies are currently used as an effective analytical tool for examining modifications in protein profiles. Understanding the natural variation of soybean seed proteins is necessary to evaluate potential unintended (collateral) effects due to transgenic modifications in genetically modified (GMO) soybeans. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to separate, identify and quantify the different classes of soybean seed proteins. Sixteen soybean genotypes, including four wild and twelve cultivated genotypes, belonging to four different subgroups were used as models for protein profile evaluation. Significant variations of allergen and anti-nutritional protein profiles were observed between two different groups, cultivated and wild soybean genotypes. However, only minor variations in protein profiles were observed within the soybean samples from the same group (cultivated or wild). These results may be useful to scientists needing to compare GMO and non-GMO soybeans once additional data are generated on additional soybean varieties and the same varieties grown at different geographical locations.