Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. For example, dimerization properties of the basic leucine zipper (bZIP) family play a critical role in regulating the unique biological functions in all eukaryotes. However, the molecular mechanism underlying the binding sequence and functional specificity of homo- versus heterodimers remains elusive. To fill this gap, we developed a double DNA Affinity Purification sequencing (dDAP-seq) technique that maps heterodimer DNA binding sites in an endogenous genome context. Our genome-wide binding profiles of twenty pairs of C/S1 bZIP heterodimers and S1 homodimers in Arabidopsis revealed that heterodimerization significantly expands the DNA binding preferences of bZIP TFs. Analysis of the heterodimer target genes in stress response and development suggest heterodimerization gives rise to regulatory responses that are distinct from the homodimers. In addition to the classic ACGT elements recognized by plant bZIPs, we found that the C/S1 heterodimers bound to motifs that might share an origin with the GCN4 cis-elements in yeast that diverged from plants more than one billion years ago. Importantly, heterodimer binding specificities can be distinguished by their relative preference for ACGT motifs versus GCN4-related motifs. More broadly, our study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation.Competing Interest StatementThe authors have declared no competing interest.