To improve our understanding of the effects of β-alanine (β) substitution and the number of heterocycles on DNA binding affinity and selectivity, we investigated the interactions of an eight-ring hairpin polyamide (PA) and two β derivatives as well as a six-heterocycle analogue with their cognate DNA sequence, 5'-TGGCTT-3'. Binding selectivity and the effects of β have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV melting, biosensor surface plasmon resonance, isothermal titration calorimetry, circular dichroism, and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔC(p) and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that β substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in the binding of PA to the mutant DNAs further confirm the position-dependent effects on the PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T·A versus A·T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of β substitution on binding tell a clear and very important story about sequence-dependent binding of PAs to DNA.