As-supplied polycrystalline ZnO samples (purity 99.9% from Sigma-Aldrich, Germany) have been annealed at different temperatures and subsequently characterized by positron annihilation spectroscopy, x-ray-diffraction (XRD) analysis, thermogravimetric analysis (TGA), and resistivity measurements. Positron annihilation lifetime analysis and coincidence Doppler-broadened electron-positron annihilation γ-radiation (CDBEPAR) line-shape measurements have been employed at a time to identify the nature of defects in differently annealed ZnO materials. Annealing up to 300 °C, an increase of defect lifetime (τ2) as well as shape parameter (S parameter) has been observed. Further annealing causes a large decrease of τ2 and S parameter. TGA study shows considerable mass loss from ZnO as the annealing temperature is increased above 300 °C. This is possibly due to oxygen evaporation from the sample. The c-axis lattice parameter, extracted from the XRD spectra, shows an increase due to annealing above 600 °C, which is a signature of the presence of a huge number of oxygen vacancies. Resistivity variations of the annealed samples are also consistent with the TG and XRD analyses. The ratio curve analysis of the CDBEPAR spectra successfully probes the change in zinc-related vacancy defects in annealed ZnO.