III-nitride heterostructure-based metal–insulator–semiconductor high-electron-mobility transistors (MIS-HEMTs), compared with Schottky and p-GaN gate HEMTs, have demonstrated significant potential in the next-generation high-power electronic devices due to their exceptional gate reliability. This study presents a comprehensive investigation of threshold voltage (VTH) instability in III-nitride heterostructure-based MIS-HEMTs, with a specific emphasis on the interfaces of the multi-heterostructures. Two widely studied amorphous materials, namely, Al2O3 and SiNx, have been extensively examined as primary gate insulators in GaN-based MIS-HEMTs. To efficiently remove native oxides from the (Al)GaN surface, a novel in situ high-temperature remote plasma pretreatment (RPP) technique has been developed. This technique involves sequential application of NH3/N2 plasmas on the (Al)GaN surface before depositing the gate insulators using plasma-enhanced atomic layer deposition. The remarkable RPP process has proven to be a highly effective method for revealing atomic steps on the GaN surface, irrespective of whether the surface has undergone oxidation or etching processes. To further enhance the interface quality and potentially reduce bulk traps in the gate insulator, optimization of deposition temperature and post-deposition annealing conditions have been explored. Additionally, an electron-blocking layer, such as SiON, is incorporated into the MIS-HEMTs to prevent electron injection into bulk traps within the insulator. Novel characterization techniques including constant-capacitance and isothermal-mode deep-level transient spectroscopy have also been developed to explore the failure mechanisms in MIS-HEMTs. These techniques allow for the differentiation between bulk traps in the GaN epitaxy and those present within the gate insulators. This in-depth physical understanding provides valuable insights into the sources of failure in GaN-based MIS-HEMTs.