Hox transcription factors direct differentiation in all tissue layers and many organ systems and therefore have highly specific tissue-dependent functions. The DNA-binding homeodomain of Hox proteins can bind a wide array of nucleotide sequences with similar high affinities, suggesting regions outside the homeodomain must impact DNA interaction. Utilizing a series of deletion mutants, we identified three binding affinity modulation regions outside the homeodomain. DNA binding is inhibited ∼2-fold by the YPWM motif and microexon regions (I1) and ∼40-fold by the large disordered I2 region. High affinity binding is partially restored by the N-terminal 174 residues (R region) in a length-dependent manner. Both I2 and R regions partially overlap the Ubx transcription activation domain, allowing communication between these functional systems. Evolutionary variations in the amino acid sequences of most of these regions potentially differentiate Ubx•DNA interactions.