In this study, structural investigations of the mismatched gallium nitride (GaN) layers grown on SiC, Al2O3 and Si substrate, were performed employing x-ray diffraction techniques. The resulted threading dislocation density evolved from the lattice mismatch, as a consequence of the strain relaxation processes, was quantified in two independent ways: firstly, using the mosaic block model, which takes into account peak width and, secondly, using the diffuse scattering model, which consider only the tails of the rocking curves. Both models indicated the close relationship between the amount of dislocations and the lattice mismatch that plays a fundamental role in TDs’ generation. To gain further insights regarding threading dislocations, grazing incidence x-ray diffraction was employed to obtain the absorption profiles of the x-rays. Although these profiles encompass the contributions of both sample absorption and dislocations, we decoupled these two effects in the framework of the dynamical theory of diffraction, showing that the existence of dislocations governs different power law dependencies for the experimental absorption profiles. Moreover, the absorption profiles bring information regarding the uniformity of the dislocations along the z-axis, revealing a uniform distribution along the z-axis for thin layer, while for the thick layer an annihilation of the dislocations was taking place. In addition, photoluminescence peaks found at 3.41 eV, 3.39 eV and 3.35 eV correspond to near band-edge (NBE) and ultraviolet donor-acceptor pair (DAP) transition. Significant changes of their intensity ratio, as well as the additional broadening of E2 (high) Raman peak confirm the presence of TDs in the investigated GaN thin films.