Allopolyploidy can result in genomic shock as divergent progenitor genomes adjust to cohabitation in the same nucleus. We are interested in how allopolypoidy affects the evolution of biosynthetic pathways, comparing natural and synthetic Nicotiana tabacum allopolyploids with their diploid progenitors. We examine how divergent flower color phenotypes and underlying pigment compositions arose in N. tabacum allopolyploids, using transcriptome analyses across flower development with a focus on differential expression of genes involved in the flavonoid biosynthetic pathway, a branched pathway that produces both anthocyanin and flavonol pigments. Differences in the composition of these pigments underlie the light pink and dark pink flower phenotypes observed in natural and synthetic N. tabacum accessions. We investigate whether reciprocal homeolog bias drives flower color differences, assess expression patterns across the flavonoid biosynthetic pathway, and correlate those patterns with pigment composition. We observe no reciprocal homeolog bias associated with flower color differences, but do find that natural allopolyploids have stronger homeolog bias than synthetic allopolyploids, suggesting the importance of trans-regulatory differences at allopolyploid origin, followed by the evolution of cis-regulatory differences between progenitor subgenomes as allopolyploids age. The maternal N. sylvestris copy of DIHYDROFLAVONOL 4-REDUCTASE (DFR), which encodes for the first enzyme in the flavonoid biosynthetic pathway that drives the flux toward anthocyanin pigments, appears to be becoming a pseudogene in natural N. tabacum accessions due to the fact that it has a frameshift, which results in the production of a nonfunctional protein. Overall expression of flavonoid biosynthetic genes correlates with flower color differences in N. tabacum allopolyploids. Specifically, a high ratio of FLAVONOL SYNTHASE (FLS), the gene encoding the enzyme that produces flavonols, to DFR, which is due to delayed activation of DFR in flower development, results in the light pink flower phenotype in N. tabacum allopolyploids. Pigment composition differences seem to involve both transcript level differences and changes in enzyme efficiency.