The nitrogen isotopic composition (15N/14N ratio, or δ15N) of enameloid-bound organic matter (δ15NEB) in shark teeth was recently developed to investigate the biogeochemistry and trophic structures (i.e., food webs) of the ancient ocean. Using δ15NEB, we present the first nitrogen isotopic evidence for trophic differences between shark taxa from a single fossil locality. We analyze the teeth of four taxa (Meristodonoides, Ptychodus, Scapanorhynchus, and Squalicorax) from the Late Cretaceous (83–84 Ma) Trussells Creek site in Alabama, USA, and compare the N isotopic findings with predictions from tooth morphology, the traditional method for inferring shark paleo-diets. Our δ15NEB data indicate two distinct trophic groups, with averages separated by 6.1 ± 2.1‰. The lower group consists of Meristodonoides and Ptychodus, and the higher group consists of Scapanorhynchus and Squalicorax (i.e., lamniforms). This δ15NEB difference indicates a 1.5 ± 0.5 trophic-level separation between the two groups, a finding that is in line with paleontological predictions of a higher trophic level for these lamniforms over Meristodonoides and Ptychodus. However, the δ15NEB of Meristodonoides is lower than suggested by tooth morphology, although consistent with mechanical tests suggesting that higher trophic-level bony fishes were not a major component of their diet. Further, δ15NEB indicates that the two sampled lamniform taxa fed at similar trophic levels despite their different inferred tooth functions. These two findings suggest that tooth morphology alone may not always be a sufficient indicator of dietary niche. The large trophic separation revealed by the δ15NEB offset leaves open the possibility that higher trophic-level lamniforms, such as those measured here, preyed upon smaller, lower trophic-level sharks like Meristodonoides. © 2024 The Authors. Geobiology published by John Wiley & Sons Ltd.