We studied single seed descent lines derived from the Kitakeiharu 717 (KKS 717) / Grandin cross. The presence of a B subunit of KKS 717-specific low-molecular-weight-glutenin subunit (LMW-GS) significantly enhanced the gluten strength. Although the C subunit showing a difference in mobility between Grandin and KKS 717 did not affect the gluten strength, the presence of this KKS 717-specific C subunit significantly decreased the gluten index in the absence of the KKS 717-specific B subunit. Using a LMW-GS locus-specific PCR primer set, we demonstrated that the B subunit of KKS 717-specific LMW-GS was encoded by the Glu-A3 locus. The use of this PCR primer set could be suitable for breeding programs aimed at improving the gluten quality.