Ion irradiation by 500 keV C+ ions has been used to introduce defects into graphene sheets deposited on SiO2 in a controlled way. The combined use of Raman spectroscopy and atomic force microscopy (AFM) allowed one to clarify the mechanisms of disorder formation in single layers, bilayers and multi-layers of graphene. The ratio between the D and G peak intensities in the Raman spectra of single layers is higher than for bilayers and multi-layers, indicating a higher amount of disorder. This cannot be only ascribed to point defects, originating from direct C+–C collisions, but also the different interactions of single layers and few layers with the substrate plays a crucial role. As demonstrated by AFM, for irradiation at fluences higher than 5 × 1013 cm−2, the morphology of single layers becomes fully conformed to that of the SiO2 substrate, i.e. graphene ripples are completely suppressed, while ripples are still …