The thermal stability of highly ordered two-dimensional superlattices consisting of dodecanethiol-ligated Au nanoparticles has been investigated using in situ grazing incidence small-angle x-ray scattering in air and in vacuum. In the lower temperature region (<70 °C), annealing in air results in a minimal change of superlattice structure, whereas annealing in vacuum leads to a considerable lattice contraction and a decrease in long-range order. At higher temperatures (>100 °C), ligand desorption causes nanocrystals to sinter locally, destroying quasi-long-range order. The sintering process is significantly enhanced in vacuum compared to the case in air due to the increased desorption rate of thiol ligands under low pressure.