Polyploidy is an important force in the evolution of flowering plants. Genomic merger and doubling induce an extensive array of genomic effects, including immediate and long-term alterations in the expression of duplicate genes (''homeologs''). Here we employed a novel high-resolution, genome-specific, mass-spectrometry technology and a well-established phylogenetic framework to investigate relative expression levels of each homeolog for 63 gene pairs in 24 tissues in naturally occurring allopolyploid cotton (Gossypium L.), a synthetic allopolyploid of the same genomic composition, and models of the diploid progenitor species. Results from a total of 2177 successful expression assays permitted us to determine the extent of expression evolution accompanying genomic merger of divergent diploid parents, genome doubling, and genomic coevolution in a common nucleus subsequent to polyploid formation. We demonstrate that 40% of homeologs are transcriptionally biased in at least one stage of cotton development, that genome merger per se has a large effect on relative expression of homeologs, and that the majority of these alterations are caused by cis-regulatory divergence between the diploid progenitors. We describe the scope of transcriptional subfunctionalization and 15 cases of probable neofunctionalization among 8 tissues. To our knowledge, this study represents the first characterization of transcriptional neofunctionalization in an allopolyploid. These results provide a novel temporal perspective on expression evolution of duplicate genomes and add to our understanding of the importance of polyploidy in plants.