Responses to allopolyploidy include unequal expression of duplicated genes, gene silencing, and sometimes genomic rearrangements. In plants, the relationship between allelic expression differences arising from changes in regulatory regions and the resulting phenotype is poorly understood because of the complexity of their genomes and lack of efficient methodology to identify regulatory variation. Identifying functionally important regulatory variation in crops such as hexaploid wheat (Triticum aestivum) is in its infancy. More knowledge is required about the contribution of participatory genomes to its transcriptome. In this paper, we demonstrate the use of allelic imbalance assays to quantify relative expression levels across tissues and growth regimes of homoeologous transcripts of the A, B, and D genomes. Polymorphisms in the type I thionins have been used as an example. We show that expression levels vary markedly and interactively over all factors. For this gene, the B genome is the smallest contributor to the transcriptome and the D genome the largest. As additional sequence information is accumulated across genomes, this assay will allow the simple study of relative expression across multiple homeologous loci.