In this paper we report on the effects of variables in thermal resistance measurement of high power GaN-based light-emitting diodes (LEDs). The investigated variables include ambient temperature, thermal interface material (TIMs) at different pressure. The combination of transient thermal measurement method and optical measurement was employed for the study. The measured thermal resistance of LED packages was found to increase with the ambient temperature. The temperature dependence of optical efficiency, forward voltage, and thermal properties of packaging materials are thought to be responsible for the increase of thermal resistance with the ambient temperature. The interface effect on the thermal resistance was studied by applying different external pressure on the interface with different TIMs. And the measured thermal resistances were found to reach stabilization at certain pressure level after initial decrease with the external applied pressure.