Rapid warming in the Arctic is drastically impacting marine ecosystems, affecting pelagic communities and food web structure. Themisto amphipods are dominant in the Arctic zooplankton community and represent a key link between secondary producers and higher trophic levels. In the Arctic seas, two coexisting species are found in high biomass: Themisto libellula, considered a true Arctic species and Themisto abyssorum, a sub-Arctic, boreal species. Many aspects of the ecology and genetic structure of these two species are not well studied, despite their importance in the food web. First, we tested both species for levels of genetic diversity and then assessed their diet spectrum with molecular methods, with a regional focus on the Greenland shelf, Fram Strait and Svalbard. Spatial genetic structure was evaluated using the mitochondrial cytochrome c oxidase subunit 1 gene (COI). Our results revealed strikingly different levels of genetic diversity: low levels in T. libellula contrasted with higher diversity in T. abyssorum. No spatial genetic structure was found, and both species exhibited high levels of connectivity and evidence of historic demographic expansion. The observed low genetic diversity, in combination with cold adaptations, could cause T. libellula to be more susceptible to the warming Arctic. In contrast, high diversity likely increases adaptive potential in T. abyssorum. In order to comprehensively characterize the prey spectrum of both Themisto species, we also applied DNA metabarcoding, also using COI, on gut contents. Both species showed a regional variation in prey spectrum. T. abyssorum’s diet showed a clear dominance of reads identified as chaetognath species, whereas T. libellula had a broader prey spectrum, including ice-associated taxa such as polar cod. Calanoid copepods did not appear as important as prey as assumed from previous (morphological) studies. Several previously overlooked jellyfish taxa were found in the stomachs of both species. The broad prey spectrum of T. libellula may indicate a higher flexibility in the light of ongoing food web changes. This work provides new data on these two ecologically important species, which can contribute to predicting how pelagic communities and species interactions will change under further climate change.