A novel 1Dx type high molecular weight glutenin subunit (HMW-GS) associated with good bread-making quality was identified in the bread wheat line W958. This glutenin subunit was designated as 1Dx5′ here for the same electrophoretic mobility as the traditional one 1Dx5 in the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. In this work, the 5′ flanking region, N-terminal as well as partial central repetitive domain of this allele was cloned and sequenced. Comparison of the amino acid sequence of 1Dx5′ with that of 1Dx5 and 1Dx2 showed the main difference being the substitution of one cysteine residue located in the central repetitive domain of 1Dx5 with serine residue in 1Dx5′ or an additional proline being inserted at the N-terminal of 1Dx5′ compared with 1Dx2. Allelic specific-polymerase chain reaction (AS-PCR) molecular markers discriminating the alleles of Glu-D1 locus were designed and applied to two F2 segregation populations. Meanwhile, the dough properties of the F2population were measured and analyzed, showing that the 1Dx5′ subunit is as good as 1Dx5 and superior than 1Dx2 in terms of dough property. The result illustrated that the inter-chain hydrogen bonds formed between the subunit repetitive domains rather than the cysteine may play the main role in bread making quality. Notably, the novel 1Dx5′ have a great portion in the F2 segregation population, suggesting the phenomenon of segregation distortion which could facilitate the introduction of 1Dx5′ into the wheat breeding program.