ObjectiveUnderstanding the spatial connectivity of elasmobranch populations is critical for regional fisheries management. The Blacktip Shark Carcharhinus limbatus is abundant in coastal waters of the Gulf of Mexico (GoM) and important in recreational and commercial fisheries. Based on genetic and tagging studies, GoM Blacktip Sharks are currently managed under separate quotas between the eastern and western GoM (divided at 88°W), but no studies have used vertebral chemistry to assess the population structure of adult Blacktip Sharks.MethodsWe compared vertebral elemental signatures (barium [Ba], magnesium [Mg], manganese [Mn], strontium [Sr], and zinc [Zn]) for the first year of life with last occupied habitats (vertebral edges) in Blacktip Sharks collected from the western (Texas and Louisiana) and eastern (Alabama and Florida) coastal waters of the northern GoM.ResultWe found significant regional differences in Ba, Mg, Mn, and Sr vertebral edge signatures, suggesting ecological separation of Blacktip Sharks. Significant correlation between first‐year and edge signatures suggested a high degree of residency between life stages. Cross‐validated discriminant function analyses yielded highest regional classification accuracies when Florida sharks were grouped separately west of 88°W (90%), demonstrating the unique elemental signatures of eastern versus western GoM Blacktip Sharks under current management delineations.ConclusionCombined, these findings demonstrate that trace element markers can distinguish regional populations of Blacktip Sharks and provide a complimentary approach in addition to genetics and physical tagging to support current stock management efforts.