The Southern Ocean, in particular the southwest Atlantic sector, is experiencing rapid environmental changes. A long-term trend of density changes of key pelagic species has been noted over the last decades: Antarctic krill populations are declining whilst salps are on the rise and shifting their distribution poleward. A similar poleward expansion is anticipated for a third key player, the hyperiid amphipod crustacean Themisto gaudichaudii, leading to an increasing overlap of the distributions of these three species. Due to major knowledge gaps in the ecology, and genetic connectivity of T. gaudichaudii, the likelihood of this shift and its consequences for the pelagic food web structure remain largely unexplored. In this context, Themisto’s genetic and trophic connectivity as well as thermal response were investigated with state-of-the-art molecular methods. Phylogeographic analyses showed genetic homogeneity between localities in the Southern Ocean and Atlantic waters combined with high degree of phenotypic plasticity enabling different lineages to thrive in regions further south. Diet analyses using DNA metabarcoding were applied to characterize regional variation in diet. These analyses showed a diet predominantly composed of krill, in particular in the Antarctic Peninsula region, showing that Themisto’s poleward range expansion can further impact the already declining krill stocks. It also unexpectedly revealed ctenophores to be an important prey, despite their reputation as “trophic dead-end”. Transcriptome analyses were used to study the thermal response of Themisto individuals from different geographic populations that were experimentally exposed to heat and cold treatments. The analysis of differentially expressed genes showed that genetic lineages differ in thermal tolerances. It also revealed a wide range of molecular mechanisms in Themisto amphipods to cope with thermal stress. These findings contribute to better predict the impact of climate-driven range shifts on the pelagic ecosystems in the Southern Ocean.