To highlight ontogenetic and geographical variability in resource utilization of Antarctic toothfish, we investigated neutral fatty acid (NFA) profiles and stable isotopes (SIs) in muscles of toothfish and their potential prey (bycatch and stomach contents), captured in fisheries in the Small Scale Research Units (SSRUs) 88.3 (the Pacific Ocean sector, POS) in austral summer during 2016–2018, making comparisons with previous dataset obtained from the Ross Sea shelf (RSS) and the Indian Ocean sector of the Antarctica during 2012–2017. We found significant differences in size distributions of regional toothfish stocks, demonstrating an ontogenetic movement into deeper water from shelf water. The relative proportions of NFAs of the IOS and POS stocks were similar to each other but differed from those of the shelf (RSS) counterpart. Although not great, isotopic differences between the shelf and slope stocks were detectable in both δ13C and δ15N values. The NFA and SI profiles show a pattern of differences between regional stocks. The NFA profiles of the RSS stock displays a considerable overlap between adults and subadults but also shows a size effect. The IOS and POS stocks showed quite large trophic overlaps, but shared very limited or no space with RSS. Adults and subadults of the RSS had overlapped trophic spaces. Sampling region, depths, and toothfish length serve as strong descriptors for most trophic markers in toothfish, emphasizing both ontogenetic and geographical variability. Finally, the regional variation in trophic niche is explained by different patterns in resource utilization, which are partitioned into two prey groups (i.e. feeding on bathypelagic vs. bathydemersal organisms), between regions and toothfish size, reflecting diet shift during ontogenetic migration across the geographic range of Antarctic waters as confirmed by the combined mixing-model calculations of both trophic markers.