Cold tolerance of adult medflies has been extensively studied but the effect of subfreezing temperatures on the immature stages remains poorly investigated, especially as far as different populations are regarded. In this study, we estimated the acute cold stress response of three geographically divergent Mediterranean fruit fly populations originating from Greece (Crete, Volos) and Croatia (Dubrovnik) by exposing immature stages (eggs, larvae, pupae) to subfreezing temperatures. We first determined the LT50 for each immature stage following one hour of exposure to different temperatures. Then eggs, larvae and pupae of the different populations were exposed to their respective LT50 for one hour (LT50 = −11 °C, LT50 = −4.4 °C, LT50 = −5 °C for eggs, larvae and pupae, respectively). Our results demonstrate that populations responded differently depending on their developmental stage. The population of Dubrovnik was the most cold-susceptible at the egg stage, whereas in that of Crete it was at the larval and pupal stage. The population of Volos was the most cold-tolerant at all developmental stages. The egg stage was the most cold-tolerant, followed by pupae and finally the 3rd instar wandering larvae. This study contributes towards understanding the cold stress response of this serious pest and provides data for important parameters that determine its successful establishment to unfavorable environments with an emphasis on range expansion to the northern, more temperate regions of Europe.