The Antarctic toothfish, Dissostichus mawsoni, is a species of Family Nototheniidae of Order Perciformes native to the Southern Ocean and one of the top predators in the Antarctic ocean. We were able to obtain 72 haplotypes (10 orders, 16 Families, 26 Genera, and 35 species) as the five-year metabarcoding analyses about the stomach contents of D. mawsoni in subarea 88.3. Based on the proportions of fish reads (99.61 %) compared with those of the cephalopods (0.27 %), D. mawsoni was piscivorous, which is consistent to the previous studies. Chinobathyscus dewitti (59.09 %), Macrourus caml (20.91%), Magnisudis prionosa (10.69 %), and Lepidonotothen squamifrons (4.52 %) turned out to be the four main preys of D. mawsoni in subarea 88.3. The metabarcoding results in subarea 88.3 were clustered together distinctly from other subarea such as 88.1 and 58.4.1. Those results strongly indicated that metabarcoding analysis of stomach contents of D. mawsoni can reflect the fish assemblage of the specimens collected. Besides regional clustering of stomach contents indicated that prey species of D. mawsoni are regionally different. Finally, the positive correlations between IRI and the proportions of NGS reads were identified with the different R values, which suggested the proportions by the metabarcoding can reflect the biomass of each prey species, albeit with a large degree of variations. For the more accurate estimation of each prey’s biomass, qPCR will be the better method, which would be further used for the long-term surveys with large research areas.