Hexagonal ZnO nanocolumns were synthesized by chemical bath deposition on previously deposited ZnO nanoseeds on Corning glass substrates, at deposition temperatures from 300 to 550 °C in steps of 50 °C, by ultrasonic spray pyrolysis technique. The ZnO nanoseeds were previously suspended in the spraying solution, with and without ultrasonic dispersion. Scanning Electron Microscopy study of surface morphology was carried out revealing the formation of hexagonal ZnO nanocolumns; the smaller diameters were obtained for ZnO seed layers deposited at substrate temperature between 400 and 450 °C. The best vertical alignment was observed on nanocolumns grown from ultrasonic-dispersed ZnO nanoseeds deposited at 400 °C. Raman spectroscopy was used to evaluate the crystal quality of the ZnO nanocolumns, a relationship was determined between nanocolumns stress with a shift to lower wavenumbers of E2H mode, the ZnO seeds deposition temperature and the nanocolumn diameters. Intrinsic defects of ZnO nanocolumns were studied with photo and cathodoluminescence spectroscopy. Also, photoluminescence life time measurements were carried out. Samples grown from ultrasonically dispersed ZnO seeds generated nanocolumns with greater stoichiometry and purity.