Using polarization sensitive two-dimensional (2D) vibrational spectroscopy on the amide I mode, the central backbone structure of trialanine in aqueous solution is investigated. We exploit the polarization sensitivity of the 2D pump-probe signal to reveal the cross-peak structure hidden under the strong diagonal peaks. The dihedral angles phi and psi characterizing the peptide backbone structure are derived directly from the cross-peak intensity and anisotropy, demonstrating the potential of 2D spectroscopy as a tool for peptide structure elucidation.