Successful development of a thermal treatment protocol depends on reliable information on fundamental thermal death kinetics of targeted insects under different heating conditions. The effects of heating rates (1, 10, and 15°Cmin −1 ), pre-treatment conditioning (30°C+6h), and the difference between long-term laboratory cultures and recently isolated cultures on thermal mortality of fifth-instar navel orangeworm, Amyelois transitella (Walker), were studied using a heating block system. There was no significant difference in insect mortality resulting from heating rates of 10 and 15°Cmin −1 . Temperature control at 1°Cmin −1 was more uniform than for the other heating rates, resulting in reduced variability for insect mortality. The mean mortality at the heating rate of 1°Cmin −1 was significantly lower than for the two faster heating rates only at 48°C+30min. The pre-treatment conditioning of fifth-instar Amyelois transitella enhanced their thermotolerance only at certain temperature–time combinations. Fifth-instars from long-term laboratory and recently isolated cultures were equally susceptible to elevated temperatures. Therefore, thermal death kinetic information obtained from the long-term laboratory cultures can be used to develop thermal protocols against field pests.