Global warming and climate change are affecting many insect species in numerous ways. These species can develop diverse mechanisms as a response to variable environmental conditions. The rise in mean and extreme temperatures due to global warming and the importance of the population’s ability to adapt to temperature stress will further increase. In this study, we investigated thermal stress response, which is considered to be one of the crucial elements of population fitness and survival in fast-changing environments. The dynamics and variation of thermal stress resistance traits in D. subobscura flies originating from two natural populations sampled from different altitudes were analysed. Three different temperature regimes (25 °C, 19 °C, and 16 °C) were used for the F1 progeny from both localities to establish six experimental groups and investigate stress resistance traits: desiccation resistance, heat knock-down resistance, starvation resistance, and chill-coma recovery time. We detected that laboratory thermal conditions and population origin may have an effect on the analysed traits, and that sex also significantly influences stress resistance. Individuals from the lower altitude reared at higher temperatures show inferior resistance to thermal shock.