The time-resolved threshold voltage instability during the circuit operation and its impact on circuit power loss are studied based on a 48–12 V synchronous hard-switching buck converter. Significantly positive Vth shift is observed along with the circuit operation time, while the ΔVth exhibits a positive correlation with the circuit operation frequency (f) and output current (Iout). The substantial ΔVth up to ~0.8 V is measured in both high-/low-side GaN HEMTs at f = 1.9 MHz and Iout = 2 A. Through the on- and off-state stress tests and HS double-pulse test, the impact of hard-switching stress on Vth instability is decoupled, while the underlying mechanisms is analyzed in detail. Moreover, the impact of Vth instability on the circuit power loss are quantitatively investigated. For instance, at f = 1.9 MHz & Iout = 2 A, the deadtime power loss significantly increased by 35.35%. The overall power loss consumed by GaN HEMTs increased up to 11.6%.