In this study we discussed crystal structure, crystallite size, lattice parameters and micro-strain, as well as defect and dislocation density of ZnO NPs are investigated using X-rays diffraction (XRD) and positron annihilation Doppler broadening energy spectroscopy (DBES). ZnO NPs size and elemental composition are studied using scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX), respectively. XRD microstructure characterization of ZnO NPs carried out using both Williamson-Hall and Rietveld methods, as well as anisotropic size-strain model (Popa rules model), is reported. The correlation between microstructural properties and DBES line-shape parameters at different chelating agent concentrations is also examined. Many defects/dislocations are observed at low chelating agent concentrations. Zn vacancies are identified as the major type of defects existing in our samples. Crystallite plot using Popa rules model revealed that crystallites at intermediate chelating agent concentrations are more isotropic, with clear spherical symmetry around optimum nucleation conditions. Good agreement between SEM estimated ZnO NPs size and XRD calculated crystallite size is observed around optimum nucleation conditions.