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Abstract Diet is a crucial trait of an animal’s lifestyle and ecology. The trophic level of an organism indicates its functional position within an ecosystem and holds significance for its ecology and evolution. Here, we demonstrate the use of zinc isotopes (δ 66 Zn) to geochemically assess the trophic level in diverse extant and extinct sharks, including the Neogene megatooth shark ( Otodus megalodon ) and the great white shark ( Carcharodon carcharias ). We reveal that dietary δ 66 Zn signatures are preserved in fossil shark tooth enameloid over deep geologic time and are robust recorders of each species’ trophic level. We observe significant δ 66 Zn differences among the Otodus and Carcharodon populations implying dietary shifts throughout the Neogene in both genera. Notably, Early Pliocene sympatric C. carcharias and O. megalodon appear to have occupied a similar mean trophic level, a finding that may hold clues to the extinction of the gigantic Neogene megatooth shark.