An atomic force microscopy (AFM) investigation into the spontaneous resolution of a P/M racemic mixture of helical self-assembled tetrarosettes 1(3)(.)(DEB)(12) (see Figure) into enantiopure domains in 2D supramolecular assemblies on highly oriented pyrolytic graphite (HOPG) is reported. These results represent the first reported case of spontaneous resolution of a racemate of chiral nanostructures.