The present work deals with self-organized TiO2 nanotube layers formed on a Ti metal substrate by anodization. For virtually any electrical or photoelectric application, these originally amorphous nanotube layers need to be converted to anatase. Most frequently, this is done by annealing at temperatures of approximately 450 °C. In the present work we use TEM and XRD to compare the annealing at a significantly lower temperature of 350 °C with that at the established 450 °C. HRTEM results show that both annealing treatments lead to comparable grain sizes in the nanotube walls; however, at 450 °C numerous defects (nanoscopic cracks) are created in the nanotube walls. Moreover, the treatment at 450 °C leads to the formation of a (rutile) oxide layer underneath the tube layers. These factors can considerably affect the overall properties of the tubes. Additionally, we point out some effects that should be carefully considered when using HRTEM investigation on TiO2 nanotubes such as e-beam-induced crystallization or tube diameter shrinkage.