The pelagic amphipods Themisto libellula and T. abyssorum represent a key zooplankton group in the polar regions. These two hyperiid amphipods are an important food source for higher trophic levels, and being carnivores, they are able to control the zooplankton standing stock in some Arctic regions. T. abyssorum is a boreal species reaching the Arctic with incoming Atlantic water and is thus likely to benefit from the ongoing Atlantification of the Arctic Ocean, whereas it may heavily impact the genuine Arctic T. libellula. Since the Arctic is undergoing drastic changes in terms of warming and sea ice loss, it is crucial to gain knowledge about the prey spectrum of these two important amphipods to predict the future of pelagic food web in a changing Arctic. In recent years, several studies were conducted concerning the diet of T. libellula and T. abyssroum. In those studies, mainly using biomarkers or stereomicroscopy, a diet consisting of the most abundant zooplankton species including calanoid copepods, euphausiids and chaetognaths, was suggested. T. abyssorum and T. libellula were also found to occupy distinct niches in the Arctic ecosystem. In this study, DNA metabarcoding was used to compare the species-level prey spectrum of the two Themisto amphipods and compare its regional variation. We found that T. libellula feeds on larvae of fish like Boreogadus saida, while this was not the case for T. abyssorum. The chaetognath Eukrohnia hamata was detected in most samples of T. abyssorum, as well as DNA of hydrozoans like Aglantha digitale or Nanomia cara, suggesting that gelatinous zooplankton represent a food source for those amphipods.