Antibiotic resistance is now one of the biggest threats humankind is facing, as highlighted in a declaration by the General Assembly of the United Nations in 2016. In particular, the growing resistance rates of Gram-negative bacteria cause increasing concerns. The occurrence of the easily transferable, plasmid-encoded mcr-1 colistin resistance gene further worsened the situation, significantly enhancing the risk of the occurrence of pan-resistant bacteria. There is therefore a strong demand for new rapid molecular diagnostic tests for the detection of mcr-1 gene-associated colistin resistance. Electrochemical impedance spectroscopy (EIS) is a well-suited method for rapid antimicrobial resistance detection as it enables rapid, label-free target detection in a cost-efficient manner. Here, we describe the development of an EIS-based mcr-1 gene detection test, including the design of mcr-1-specific peptide nucleic acid probes and assay specificity optimization through temperature-controlled real-time kinetic EIS measurements. A new flow cell measurement setup enabled for the first time detailed real-time, kinetic temperature-controlled hybridization and dehybridization studies of EIS-based nucleic acid biosensors. The temperature-controlled EIS setup allowed single-nucleotide polymorphism discrimination. Target hybridization at 60 °C enhanced the perfect match/mismatch (PM/MM) discrimination ratio from 2.1 at room temperature to 3.4. A hybridization and washing temperature of 55 °C further increased the PM/MM discrimination ratio to 5.7 by diminishing the mismatch signal during the washing step while keeping the perfect match signal. This newly developed mcr-1 gene detection test enabled the direct, specific label, and amplification-free detection of mcr-1 gene harboring plasmids from Escherichia coli.