The performance of AlGaN/GaN High Electron Mobility (HEMT) transistors is directly related to the electrical characteristics of the two-dimensional electron gas formed at the interface thanks to the piezoelectric field. Modification of the Al content or thickness of the AlGaN layer can within a certain limit modify the carrier density and mobility in the 2DEG. However, further reduction of the sheet resistance requires strain engineering of the heterostructure. An effective way to reduce the sheet resistance, as well as to lower the threading dislocation (TD) density, is to perform strain engineering through the use of low temperature AlN interlayers inserted in the GaN buffer layer. From correlation of AFM, TEM and HRXRD mapping of the HEMT layers, the strain modification, as well as the mechanism reducing the TD density, can be explained by the highly defected nature of the AlN interlayer grown at low temperature, as well as its very small thickness. The LT AlN acts as a second nucleation layer for the GaN grown on top. Contrarily, when the AlN interlayer is grown at 1050°C, its high crystalline quality and the possibility to grow pseudomorphic and abrupt interfaces, allows its use at the AlGaN/GaN interface. Optimal combination of the AlGaN and AlN layer thickness leads to record values of the mobility at room temperature of 2050 cm2/Vs, for heterostructures grown on sapphire, which is approaching state-of-the-art for HEMT grown on SiC.