DNA is a re-configurable, biological information-storage unit, and much remains to be learned about its heterogeneous structural dynamics. For example, while it is known that molecular dyes templated onto DNA exhibit increased photostability, the mechanism by which the structural dynamics of DNA affect the dye photophysics remains unknown. Here, we use femtosecond, two-dimensional electronic spectroscopy measurements of a cyanine dye, Cy5, to probe local conformations in samples of single-stranded DNA (ssDNA–Cy5), double-stranded DNA (dsDNA–Cy5), and Holliday junction DNA (HJ–DNA–Cy5). A line shape analysis of the 2D spectra reveals a strong excitation–emission correlation present in only the dsDNA–Cy5 complex, which is a signature of inhomogeneous broadening. Molecular dynamics simulations support the conclusion that this inhomogeneous broadening arises from a nearly degenerate conformer found only in the dsDNA–Cy5 complex. These insights will support future studies on DNA’s structural heterogeneity.