Understanding the role of defects in graphene is the key to tailoring the properties of graphene and promoting the development of graphene-based devices. Defects can affect the electronic properties of a device while also offering a means by which to functionalize the local properties. Using tip-enhanced Raman spectroscopy (TERS), heightened defect sensitivity was demonstrated on graphene edges, folds, and overlapping regions. Measurements confirm that TERS can provide simultaneous structural and spectral information on a localized scale, hence offering defect characterization on a scale that is not obtainable using conventional Raman spectroscopy. This study observed preferential enhancement of the D band signal on multilayered graphene and ultrathin graphite; in addition, other key defect signatures were also enhanced and detected. We present our findings in relation to theoretical predictions of graphene defect signatures and an analysis of the sensitivity of TERS in measuring two-dimensional structures. Copyright © 2013 John Wiley & Sons, Ltd.