Gene expression is a complex process, requiring precise spatial and temporal regulation of transcription factor activity; however, modifications of individual cis- and trans-acting modules can be molded by natural selection to create a sizeable number of novel phenotypes. Results from decades of research indicate that developmental and phenotypic divergence among eukaryotic organisms is driven primarily by variation in levels of gene expression that are dictated by mutations, either in structural or regulatory regions, of genes. The relative contributions and interplay of cis- and trans-acting regulatory factors to this evolutionary process, however, remain poorly understood. Analysis of eight genes in the Bz1–Sh1 interval of Zea mays (maize) indicates significant allele-specific expression biases in at least one tissue for all genes, ranging from 1.3-fold to 36-fold. All detected effects were cis-regulatory in nature, although genetic background may also influence the level of expression bias and tissue specificity for some allelic combinations. Most allelic pairs exhibited the same direction and approximate intensity of bias across all four tissues; however, a subset of allelic pairs show alternating dominance across different tissue types or variation in the degree of bias in different tissues. In addition, the genes showing the most striking levels of allelic bias co-localize with a previously described recombination hotspot in this region, suggesting a naturally occurring genetic mechanism for creating regulatory variability for a subset of plant genes that may ultimately lead to evolutionary diversification.