The δ15N values of organisms are commonly used across diverse ecosystems to estimate trophic position and infer trophic connectivity. We undertook a novel cross-basin comparison of trophic position in two ecologically well-characterized and different groups of dominant mid-water fish consumers using amino acid nitrogen isotope compositions. We found that trophic positions estimated from the δ15N values of individual amino acids are nearly uniform within both families of these fishes across five global regions despite great variability in bulk tissue δ15N values. Regional differences in the δ15N values of phenylalanine confirmed that bulk tissue δ15N values reflect region-specific water mass biogeochemistry controlling δ15N values at the base of the food web. Trophic positions calculated from amino acid isotopic analyses (AA-TP) for lanternfishes (family Myctophidae) (AA-TP ,2.9) largely align with expectations from stomach content studies (TP ,3.2), while AA-TPs for dragonfishes (family Stomiidae) (AA-TP ,3.2) were lower than TPs derived from stomach content studies (TP,4.1). We demonstrate that amino acid nitrogen isotope analysis can overcome shortcomings of bulk tissue isotope analysis across biogeochemically distinct systems to provide globally comparative information regarding marine food web structure.