Soybean [Glycine max (L.) Merr.,] is considered a high quality source of oil and protein for food and feed. However, the several antinutritional factors ( lipoxygenase, trypsin inhibitor, lectin, and P34 allergen protein) present in raw mature soybean seeds. Soybean Kunitz trypsin inhibitor (KTI) protein has been proposed as one of the major antinutritional factor (Westfall and Hauge, 1948). KTI protein is a small, monomeric and non-glycosylated protein containing 181 amino acid residues. This 21.5 kDa non-glycosylated protein was first isolated and crystallized from soybean seeds by Kunitz (1945). KTI protein can cause the induction of pancreatic enzyme hypersecretion and a fast stimulation of pancreas growth, which is histologically described as pancreatic hypertrophy and hyperplasia (Liencer, 1995). Also, KTI may cause unfavorable physiological effects (Vasconcelos et al., 2001) and decrease weight gain in animals (Palacios et al., 2004). Proper heat processing is required to destroy KTI protein. However, excessive heat treatment may lower amino acid availability. The genetic removal of the KTI protein will improve the nutritional value of soybean. From the USDA germplasm collection, two soybean accessions (PI157440 and PI196168) lacking the KTI protein have been identified (Orf and Hymowitz, 1979). Based on the availability of soybean null lines lacking the KTI protein, it was suggested that KTI protein is not essential for soybean growth or development. Five electrophoretic forms of KTI have been discovered. The genetic control of four forms, Ti a, Ti b, Ti c, and Ti d, has been reported as a codominant multiple allelic series at a single locus (Singh et al., 1969; Hymowitz and Hadley, 1972; Orf and Hymowitz, 1979). Orf and Hymowitz (1979) found that the fifth form does not exhibit a soybean trypsin inhibitor-A2 band and is inherited as a recessive allele designated ti. Studies of amino acid and nucleotide sequences of polymorphic variants of KTI have revealed that there is a large sequence differences in nine amino acid residues between Ti a and Ti b (Song et al., 1993; Wang et al., 2004). Each Ti c, Ti d and Ti e differ by only one amino acid from Ti a type and Ti f differs by one amino acid from Ti b type (Wang et al., 2004). The Ti locus has been located on linkage group 9 in the classical linkage map of soybean (Hildebrand et al., 1980; Kiang, 1987), which is integrated in molecular linkage map A2 (chromosome number 8) of the USDA/Iowa State University soybean molecular linkage map (Cregan et al., 1999).