Technical Abstract: Traditional views on the potential genetic effects of polyploidy in allohexaploid wheat (Triticum aestivum L.) have primarily emphasized aspects of greater coding sequence variation and the enhanced potential to acquire new gene functions through mutation of redundant loci. The extent and significance of regulatory variation has been relatively unexplored. Recent investigations suggest that differential expression of homoeologous transcripts is common in wheat. In order to establish a timeline for such regulatory changes and estimate the frequency of non-additive expression of homoeologous transcripts in newly-formed T. aestivum, gene expression was characterized in a synthetic T. aestivum line and T. turgidum and Aegilops tauschii parents. Applying cDNA-SSCP analysis to study 30 arbitrarily-selected homoeologous transcripts revealed that four (~13%) showed differential expression of homoeoalleles in seedling leaf tissue of synthetic T. aestivum. In microarray expression experiments, synthetic T. aestivum gene expression was compared to mid-parent expression model values calculated from parental T. turgidum and Ae. tauschii expression levels. Approximately 16% of genes were estimated to display non-additive expression in synthetic T. aestivum. Homoeolog expression patterns of non-additive transcripts identified by microarrays suggest that cis-acting regulatory variation is often responsible for non-additive gene expression levels. These results demonstrate that allopolyploidization, per se, results in rapid initiation of differential expression of homoeologous loci and non-additive gene expression in T. aestivum.