Growing interest in the Alligator Gar Atractosteus spatula among anglers and fishery managers has inspired efforts to better manage populations. Successful management requires identifying population structure and understanding the distribution of stocks and associated differences in life history. This is particularly important in river systems along the coast of the Gulf of Mexico, where transitions from freshwater rivers to saltwater bays provide the potential for life history diversification. We used otolith microchemistry and genetics to assess population structure of Alligator Gars in the Guadalupe River–San Antonio Bay system, Texas. Lifetime Sr:Ca revealed three, distinct life histories that differed in prevalence across the system. River-resident fish (i.e., fish exclusive to freshwater) were present throughout the river but were most common in the uppermost river reach (74% of upper river fish). Transient fish that used both river and bay habitats were also found throughout the river but were most prevalent in the lowermost river reach (66% of lower river fish) and bay (91% of bay fish). Bay residents (i.e., fish exclusive to salt water) were detected but comprised only 9% of bay fish. Haplotype diversity based on mitochondrial DNA was lowest in the upper river, indicating limited gene flow compared with the lower river and bay. Similarly, nuclear DNA analyses indicated nonrandom mating between fish from the upper river, lower river, and bay. The differences in Alligator Gar movement and genetics along the river–bay continuum suggest the presence of a river resident stock that predominates the upper river, and a transient stock that predominates the lower river and bay. Therefore, a local-scale management approach, consistent with the spatial partitioning between stocks, would conserve life history and genetic diversity within the system and provide opportunities to meet the needs of a diverse angling constituency. Understanding how population dynamics differ between stocks is needed to develop appropriate fishery management objectives and corresponding regulations for Alligator Gar.