1Bx14 is a member of the high molecular weight (HMW) glutenin subunits specified by wheat Glu-B1-1 alleles. In this work, we found that the full-length amino acid sequence of 1Bx14 derived from cloned coding region was similar, but not identical, to that of 1Bx20. In the N-terminal domains of 1Bx14 and 1Bx20, the last two of the three cysteine residues, which are conserved in 1Bx7, 1Bx17 and homoeologous 1Ax and 1Dx subunits, were replaced by tyrosine residues. In the 5′ flanking regions (-900 to -1,200 bp relative to the start codon), a novel miniature inverted-repeat transposable element insertion was present in 1Bx14 and 1Bx20 but not 1Bx7 and 1Bx17. 1Bx14 and 1Bx20 like alleles were readily found in tetraploid wheat subspecies but not several S genome containing Aegilops species. Phylogenetic analysis showed that the four molecularly characterized Glu-B1-1 alleles (1Bx7, 1Bx14, 1Bx17, 1Bx20) could be divided into two allelic lineages. The lineage represented by 1Bx7 and 1Bx17 was more ancient than the one represented by 1Bx14 and 1Bx20. Combined, our data establish that 1Bx14 and 1Bx20 represent a novel subclass of Glu-B1-1 alleles. Based on current knowledge, potential mechanism involved in the differentiation of two Glu-B1-1 lineages is discussed.