In this study, density functional theory (DFT) and time dependent density functional theory (TD-DFT) are used to investigate the stabilities and spectral properties [IR, UV–vis, and two-photon absorption (2PA)] of two sets of modified RNA nucleobase tautomers. The modifications introduce either a sulfur or selenium atom to form an isothiazolo[4,3-d]pyrimidine or isoselenazolo[4,3-d]pyrimidine heterocyclic core, respectively. The relative stabilities of both sets of modified tautomers determined with B3LYP/6-31++G(d,p) reveal that in water (with a polarizable continuum model), the 6-keto-2-amino tautomer of guanine and the rare 4-imino-2-keto tautomer of cytosine may be present at significant populations, whereas the 6-enol-2-amino tautomer of guanine is more common in the gas phase. The identification of these modified tautomers is possible due to the natural differences in their vibrational modes and hence IR spectra. Furthermore, the photophysical properties of both these sets of modified tautomers indicate that excitation and emission energies are shifted relative to their more abundant form in both one photon absorption and emission and 2PA spectra, as determined at the B3LYP/6-31++G(d,p) and CAM-B3LYP/aug-cc-pVDZ levels of theory, respectively. Even though the 2PA cross sections in water for all of the species are small (0.3–2.3 GM), the modified cytosine tautomer shows promise, as its cross section is larger than the more dominant form. The spectral separation between the dominant form and the tautomers of isoselenazole and isothiazole modified cytosine and guanine are relatively similar, suggesting both modifications could be useful in elucidating the tautomers from their more abundant counterparts.