Wheat prolamin-box binding factor (WPBF) was shown to be an activator of Triticum aestivum L. storage protein genes. Three homoeologous genes encoding this transcription factor were isolated from a bacterial artificial chromosome genomic library and sequenced. The genes all have two exons separated by an intron of approximately 1,000 bp where the second exon contains the entire coding sequence. Many differences were found between homoeologous sequences, but none of them is predicted to significantly alter the sequence of the putative encoded protein. The three homoeologous genes are specifically expressed in grain from 3 to 39 days after anthesis. The allelic variation of a genetically diverse collection of 27 bread wheat lines was assessed. One, five, and one single-nucleotide polymorphisms (SNPs) were detected in the wPbf genes for the A, B, and D genomes, respectively. Physical and genetic mapping utilizing some of the SNPs identified confirmed that wPbf genes are located close to the centromeres on the homoeologous group 5 chromosomes. The low level of allelic diversity found in wPbf genes may suggest that these genes play a key role and are thus constrained by selection.