Atomic-scale defects in graphene layers alter the physical and chemical properties of carbon nanostructures. Theoretical predictions have recently shown that energetic particles such as electrons and ions can induce polymorphic atomic defects in graphene layers as a result of knock-on atom displacements. However, the number of experimental reports on these defects is limited. The graphite network in single-walled carbon nanotubes has been visualized by transmission electron microscopy (TEM) and their chiral indices have been determined. But the methods used require a long image acquisition time and intensive numerical treatments after observations to find an 'average' image, which prevents the accurate detection and investigation of defect structures. Here we report observations in situ of defect formation in single graphene layers by high-resolution TEM. The observed structures are expected to be of use when engineering the properties of carbon nanostructures for specific device applications.