Pelagic hyperiid amphipods represent an important trophic link between the herbivore-omnivore zooplankton community and various higher trophic levels. The two pelagic hyperiid amphipods, Themisto gaudichaudii and Vibilia armata, are abundant in the southern Benguela upwelling system. T. gaudichaudii is recognized as a free-swimming species, while V. armata has shown an association with gelatinous zooplankton, particularly during their juvenile stages. So far, morphological identification of prey remains in the stomach contents has been commonly performed to reveal the food items in the diet of hyperiid species. But the stomach content examination technique often overlooked the highly degraded, fragile, and fragmented prey organisms (e.g., gelatinous zooplankton). Thus, the dietary spectra of both species remain poorly understood, especially in the Benguela upwelling system. DNA metabarcoding is an emerging and powerful molecular technique that can be used to investigate diet compositions using DNA fragments available in stomachs or feces. This molecular approach allows prey identification at a high taxonomic resolution to species or genus level, depending on the completeness of the reference database. In this study, we apply DNA metabarcoding to identify the prey composition in the stomachs of T. gaudichaudii and V. armata, targeting the mitochondrial cytochrome c-oxidase subunit 1 (COI) and the nuclear 18S rDNA regions. Further, we assess variations of prey compositions among the different size categories of each species and different localities in the southern Benguela upwelling system. The outcome of this study provides an insight into the trophic status of the two hyperiids and ultimately contributes to a comprehensive understanding of their ecological role in the southern Benguela food web.