Polyploidization is a driving force of wheat (Triticum aestivum) evolution and speciation, yet its impact on epigenetic regulation and gene expression remains unclear. Here, we constructed a high-resolution epigenetic landscape across leaves, spikes, and roots of hexaploid wheat and its tetraploid and diploid relatives. Inter-species stably expressed genes exhibited conserved amino acid sequences under strong purifying selection, while dynamically expressed genes were linked to species-specific adaptation. During hexaploidization, dominant D-subgenome homoeolog expression was suppressed via reduced activating epigenetic signals, converging expression with the A and B subgenomes. Proximal chromatin regions near genes were more stable, whereas distal regions, particularly distal cis-regulatory elements (dCREs) regulated by H3K27ac and H3K4me3, exhibited higher dynamism. Sequence variations in these dCREs led to differential gene regulation, influencing traits such as spike development. For instance, the two haplotypes of the DENSE AND ERECT PANICLE (TaDEP-B1) dCRE region resulted in significant differences in its expression and spikelet numbers. We also observed a coevolution of transcription factors and their binding sites, particularly within the expanded ERF family, which regulates spike morphology. This study highlights the interplay between sequence variation and epigenetic modifications in shaping transcriptional regulation during wheat speciation, offering valuable insights for genetic improvement.