In this study, we investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), the cultivated soybean (G. max) ancestors of modern N. American soybean cultivars (old), modern N. American soybean (elite), and Asian cultivated soybean landraces that were the immediate results of domestication from the wild soybean. Proteins were well separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and stained protein cut from a 2D-PAGE indicated that KTI exists as multiple isoforms (spots) in soybean. Protein spots of KTI were identified and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although overall distribution patterns of the KTI protein spots appeared similar, the number and intensity of the protein spots between wild and cultivated genotypes varied. Three KTI peptides were identified in three of the wild genotypes, PI 393551, PI 407027 and PI 407282, in which KTI3 peptide showed highest intensity. The remaining wild genotype, PI 366120, showed four protein spots. In contrast, the ancestors, modern and Asian landrace genotypes showed only two protein spots corresponding to KTI. On the basis of DNA blot analysis, there is one copy of the KTI3 gene in all 16 genotypes. Polymorphism was detected in one of the wild genotypes (PI 366120) both in proteomic and genomic analyses. Our data suggest that the major variation of protein profiles were between wild and cultivated soybean genotypes rather than among genotypes in the same group. Genetic variation of KTI1, KTI2 and KTI3-related genes were detected within and between groups.