Extensive studies combining optical and calorimetric measurements sensitive to the global melting temperature of a duplex with sequences that systematically varied the nucleobase ordering established our current understanding of duplex thermodynamics and secondary structure. However, this foundational data still provides only an indirect measurement of the contribution of individual structural elements to the total stability of the duplexed molecules. The most widely used method to monitor populations of single and double-stranded DNA as a function of temperature (Tm) is UV-visible spectroscopy, 108 as DNA duplexes have a UV-vis absorption at 260 nm that increased in intensity when single-stranded as compared to duplexed forms. It is thus an excellent measurement of melting of entire strands but cannot report on local structure that underpins the variability in sequence global Tm, well-known to be provided by stacking from nearest neighbor interactions. To provide more accurate measurements of thermodynamic increments that must be overcome during single base pair melting to access the genetic code, a sub-molecular scale thermometer (TmL) is required for measuring local stability in the duplex structure. Here, we build on our recently established nucleobase isotope editing methods using the T 13 C editing and apply this as a sub-molecular scale probe of the transition temperatures at individual base pairs in short DNA duplexes. We do this by developing a unique, spatially defined, and potentially quite powerful variable temperature isotope edited IR method to measure the signals from the isotopically labeled position as a function of temperature.