Transmission electron microscopy (TEM) based orientation mapping has been used to measure the length fraction of coherent and incoherent Σ3 grain boundaries in a series of six nanocrystalline Cu thin films with thicknesses in the range of 26–111 nm and grain sizes from 51 to 315 nm. The films were annealed at the same temperature (600 °C) for the same length of time (30 min), have random texture, and vary only in grain size and film thickness. A strong grain size dependence of Σ3 (coherent and incoherent) and coherent Σ3 boundary fraction was observed. The experimental results are quantitatively compared with three physical models for the formation of annealing twins developed for microscale materials. The experimental results for the nanoscale Cu films are found to be in good agreement with the two microscale models that explain twin formation as a growth accident process.