Hall effect measurements on unintentionally doped Al0.25Ga0.75N/GaN/AlN heterostructures grown by metal organic chemical vapor deposition (MOCVD) were carried out as a function of temperature (20–300 K) and magnetic field (0–1.4 T). Magnetic-field-dependent Hall data are analyzed using the quantitative mobility spectrum analysis (QMSA) technique. The QMSA technique successfully separated electrons in the 2D electron gas (2DEG) at the Al0.25Ga0.75N/GaN interface from other 2D and 3D conduction mechanisms of the samples. 2DEG mobilities, carrier densities and conductivities of the investigated samples are compared at room temperature and low temperature (20 K). For a detailed investigation of the 2DEG-related growth parameters, the scattering analyses of the extracted 2DEG were carried out for all of the samples. Using the results of the scattering analyses, the relation between the growth and scattering parameters was investigated. Increments in the interface roughness (IFR) are reported with the increased GaN buffer growth temperatures. In addition, a linear relation between the deformation potential and interface roughness (IFR) scattering is pointed out for the investigated samples, which may lead to a better understanding of the mechanism of IFR scattering.