The key feature for the precise tuning of Vth in GaN-based metal-insulator-semiconductor (MIS) high electron mobility transistors is the control of the positive fixed charge (Qf) at the insulator/III-N interfaces, whose amount is often comparable to the negative surface polarization charge (Qpol−). In order to clarify the origin of Qf, we carried out a comprehensive capacitance-voltage (C-V) characterization of SiO2/AlxGa1–xN/GaN and SiN/AlxGa1–xN/GaN structures with Al composition (x) varying from 0.15 to 0.4. For both types of structures, we observed a significant Vth shift in C-V curves towards the positive gate voltage with increasing x. On the contrary, the Schottky gate structures exhibited Vth shift towards the more negative biases. From the numerical simulations of C-V curves using the Poisson's equation supported by the analytical calculations of Vth, we showed that the Vth shift in the examined MIS structures is due to a significant decrease in the positive Qf with rising x. Finally, we examined this result with respect to various hypotheses developed in the literature to explain the origin of the positive Qf at insulator/III-N interfaces.