In this paper, the effects of thickness of AlN nucleation layer grown at high temperature on AlN epi-layer crystalline quality are investigated. Crack-ftee AlN samples with various nucleation thicknesses are grown on sapphire substrates by plasma-assisted molecular beam epitaxy. The AlN crystalline quality is analysed by transmission electron microscope and x-ray diffraction (XRD) rocking curves in both (002) and (102) planes. The surface profiles of nucleation layer with different thicknesses after in-situ annealing are also analysed by atomic force microscope. A critical nucleation thickness for realising high quality AlN films is found. When the nucleation thickness is above a certain value, the (102) XRD full width at half maximum (FWHM) of AlN bulk increases with nucleation thickness increasing, whereas the (002) XRD FWHM shows an opposite trend. These phenomena can be attributed to the characteristics of nucleation islands and the evolution of crystal grains during AlN main layer growth.