Measuring the strength of the hydrogen bonds between DNA base pairs is of vital importance for understanding how our genetic code is physically accessed and recognized in cells, particularly during replication and transcription. Therefore, it is important to develop probes for these key hydrogen bonds (H-bonds) that dictate events critical to cellular function, such as the localized melting of DNA. The vibrations of carbonyl bonds are well-known probes of their H-bonding environment, and their signals can be observed with infrared (IR) spectroscopy. Yet, pinpointing a single bond of interest in the complex IR spectrum of DNA is challenging due to the large number of carbonyl signals that overlap with each other. Here, we develop a method using isotope editing and infrared (IR) spectroscopy to isolate IR signals from the thymine (T) C2═O carbonyl. We use solvatochromatic studies to show that the TC2═O signal's position in the IR spectrum is sensitive to the H-bonding capacity of the solvent. Our results indicate that C2═O of a single T base within DNA duplexes experiences weak H-bonding interactions. This finding is consistent with the existence of a third, noncanonical CH···O H-bond between adenine and thymine in both Watson-Crick and Hoogsteen base pairs in DNA.