Recently, several families of small-molecule ligands have been developed to selectively target DNA pairing defects, such as abasic sites and mismatched base pairs, with the aim to interfere with the DNA repair and the template function of the DNA. However, the affinity and selectivity (with respect to well-matched DNA) of these ligands has barely been evaluated in a systematic way. Herein, we report a comparative study of binding affinity and selectivity of a representative panel of 16 ligands targeting abasic sites and a T-T mismatch in DNA, using a fluorescence-monitored melting assay. We demonstrate that bisintercalator-type macrocyclic ligands are characterized by moderate affinity but exceptionally high selectivity with respect to well-matched DNA, whereas other reported ligands show either modest selectivity or rather low affinity in identical conditions.