While there are a number of electrochemical methods reported that enable the detection of single nucleotide mismatches, the determination of mismatch position in a double stranded DNA remains an unsolved challenge. Using a model system, we systematically explored the electrochemical response of all possible positions of single nucleotide mismatches in a set of 25-mer DNA films. These ds-DNA sequences each with a single mismatch at one of the twenty-five positions were bound to gold surfaces through a Au–S linkage and analyzed by electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) in the absence and presence of Zn²⁺. We expected a unique response from each mismatched sequence in order to discriminate the mismatch positions. A pattern emerges between the electrochemical signals and mismatch positions. The positions can be grouped broadly into positions that exhibit large differences between matched and mismatched DNA (around positions 5 and 9) and those that exhibit smaller differences (around positions 1, 13 and 23) in the charge transfer resistance ΔR_ct, evaluated by EIS, and the apparent rate constant k⁰, evaluated by SECM. To the best of our knowledge, this is the first study evaluating the electrochemical response of a single nucleotide mismatch as a function of mismatch positions along an oligonucleotide sequence.