Understanding population genetic structure and patterns of gene flow is important for effective decision making and the preservation of genetic diversity, especially when managing protected species. Historically, Gulf sturgeon have been managed by river system, with early evidence supporting spatially distinct genetic structure across 7 natal populations. However, an increasing number of studies recognize that some rivers possess 2 genetically distinct sturgeon stocks residing within the same drainage. This, combined with evidence of both spring and fall spawning events within these drainages, suggests the distinction between these stocks may be temporally mediated, as similarly exhibited by the closely related Atlantic sturgeon. We analyzed the movements of 136 Gulf sturgeon within the Suwannee River, Florida, USA, between 2008 and 2022 using acoustic telemetry. We assessed migratory patterns and presence within known spawning habitat to categorize individuals as exhibiting either spring, fall, or unknown spawning behaviors each year. These individuals were also genotyped for 14 microsatellite loci to assign them to a genetic group. Our results reinforce previous work suggesting the presence of 2 genetically distinct stocks in the Suwannee River and indicate this genetic structure is driven by fidelity to temporally distinct spawning events, with one stock spawning in the spring and the other in fall. The delineation of these 2 sympatric stocks, historically managed as one, has management implications for the recovery of this species.