The influence of temperature on the frictional properties and molecular structure of hexadecanethiol self-assembled monolayers (SAMs) adsorbed on gold has been measured by atomic force microscopy (AFM) in a vacuum environment. The frictional response of hexadecane thiol films decreases significantly when the as-deposited SAM film is heated from room temperature to 330 K, with a corresponding increase in surface order. The changes observed during the first heat treatment are irreversible, with the room-temperature frictional response lowered by approximately a factor of 4. However, subsequent heating cycles produce a reversible change in interfacial friction for temperatures up to 350 K, with interfacial friction increasing with increasing temperature. Further heating the SAM film above 370 K produces a significant and irreversible increase in friction. At 400 K, the lattice-resolved structure of the Au(111) surface is observed, indicating the instability and initial stages of desorption of the alkanethiol film at this temperature. Following surface anneals to 500 K, only small three-dimensional islands of residual thiol are observed in large-scale topographic images and the frictional properties largely reflect those of bare gold. The reversible increase in the frictional properties of the hexadecanethiol film with increasing temperature is ascribed to a decrease in the molecular order and the effective density of the film. Above the temperature threshold for film damage, the irreversible increase in frictional response is ascribed to energy being dissipated through ploughing and displacement of the film.