Anodic oxidation of titanium substrates produces TiO2 nanotubes, a potential surface coating for biomedical implants. Though the influence of voltage, electrolyte composition, and anodization time on the nanostructure is largely understood, little is known regarding the influence of substrate preparation. This study reports the effect of metallographic preparation on nanotube structure characteristics. Titanium substrates were prepared using three techniques. Half the mechanical specimens underwent extensive SiC grinding and 0.5 μm colloidal silica H2O2 polishing; half used fewer steps: 600 SiC, 9 μm suspension, and colloidal silica H2O2 polish. The electrolytic polishing used two electrolytes, one with ZnCl2 and AlCl3, and one with perchloric acid, only the latter producing specimens suitable for nanotube growth. An ammonium fluoride and ethlyene glycol electrolyte was used to anodized the substrates. The nanotubes were characterized using SEM, the results correlated with the polishing techniques. Nanotubes from the mechanical and electrolytic substrates showed differences in geometry.