We have used in situ low-energy electron microscopy (LEEM) to correlate the atomic and electronic structure of graphene films on polycrystalline Ni with nm-scale spatial resolution. Spatially resolved electron scattering measurements show that graphene monolayers formed by carbon segregation do not support the π-plasmon of graphene, indicating strong covalent bonding to the Ni. Graphene bilayers have the Bernal stacking characteristic of graphite and show the expected plasmon loss at 6.5 eV. The experimental results, in agreement with first-principles calculations, show that the π-band structure of free-standing graphene appears only in films with a thickness of at least two layers and demonstrate the sensitivity of the plasmon loss to the electronic structure. © 2010 American Chemical Society.