This study comprehensively investigates the threshold voltage (Vth) instability of AlGaN/GaN MIS-HEMTs under semi-on state conditions. The effects of various temperatures and drain biases are studied. Under semi-on bias conditions, a significant negative Vth shift up to −1.0 V is observed in MIS-HEMTs with 10-nm in-situ SiN gate dielectrics, whereas Schottky HEMTs exhibit a negligible Vth shift. A gate current analysis, comparing the MIS-HEMT with the source grounded versus floating, suggests that hot electrons induce generation of holes under the semi-on state. TCAD simulations corroborate that the high electric field across the channel of the MIS-HEMT induce hot electrons and hole generation under the semi-on state; dynamic interaction of hot electrons and holes with the gate SiN/AlGaN interfacial states leads to bias temperature instability (BTI) of the Vth of the MIS-HEMT. These findings suggest that optimizing the gate dielectric and its interface with the top barrier is crucial to improve the semi-on state stability of radio-frequency (RF) MIS-HEMTs.