Multiple forms of DNA damages such as base modifications, double-strand breaks, and mispairings are related to inheritable diseases, cancer, and aging. Here, the structural changes of duplex DNA upon incorporation of mismatched base pairs are examined by EPR spectroscopy. Two ethynyl-7-deaza-2'-deoxyadenosine residues separated by two nucleotides were incorporated in DNA and functionalized with 4-azido-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-azido TEMPO) by the click reaction. Mismatches such as dT.dT or dA.dA mispairs were positioned between these two spin labels in DNA duplexes. Pulse EPR experiments reveal that the mismatch-induced local conformational changes are transmitted to the flanking nucleotides and that the impact of this mismatch depends on the nearest neighbor environment.