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Three low molecular weight (LMW) glutenin subunit genes were isolated from hexaploid wheat cultivars (Triticum aestivum. L) using LMW-GS specific primers and designated as Jokyung II-2, CS III-5 and Keumkang 6-12, respectively. All sequences were classified as LMW-i type genes based on the presence of an N-terminal isoleucine residue. All comprised of four regions, the signal peptide, N- and C- terminal domains, and a repetitive domain. Although they showed high similarity with other LMW-i type genes from hexaploid bread wheat, they also displayed unique features. Particularly, Keumkang 6-12 subunit contained an extra cysteine residue in the C-terminus except for eight conserved cysteines, which resulted from a single-nucleotide polymorphism (SNP) of the A–G transition, namely tyrosine-cysteine substitution at position 335 from the N-terminal end. In addition, a total of 15 SNPs and one insertions/deletions (InDels) were detected among Jokyung II-2, CS III-5 and Jokyung HQ6199333 and 10 other LMW-i genes. Two dimensional gel electrophoresis and LC-ESI MS/MS analysis revealed that LMW i-type glutenin subunit of the Jokyung is Glu-A3c. The phylogenic analysis showed that LMW i-type genes were divided roughly into diploid and hexaploid groups and the hexaploid were further classified into 7 subgroups (Glu-A3a~g). We also found length polymorphism in the repetitive domain showing 2~179 amino acid residues variation among the previously published LMW-i type glutenin subunits. The length polymorphism can be used as reliable genetic marker for use in marker-assisted breeding to select for specific alleles of the quality-determining locus in wheat breeding program.