At one time, adult Atlantic sturgeon supported a signature fishery in the Hudson River and many other major rivers coastwide. Overharvest and several other anthropogenic stressors led to a reduction of abundance of almost all populations, state and federal harvest moratoriums, and subsequent listing of the species as five Distinct Population Segments under the U.S. Endangered Species Act. Although Atlantic sturgeon exhibit strong spawning fidelity to their natal rivers, subadults and adults are highly migratory for prolonged periods in coastal waters and are seasonally found in non-natal estuaries. DNA approaches are the “gold standard” to distinguish populations and to identify the river-of-origin of individuals outside of their natal estuaries. Similarly, use of acoustic telemetry has been adopted by many sturgeon researchers to better understand their complex and prolonged migratory behavior within and outside of their natal estuaries. In this study, we married the two approaches to evaluate the veracity of the DNA approaches in determining the river-of-origin of sturgeon by comparing genetic assignments to individual spawning rivers and DPS with the actual detection of acoustically tagged fish in the Hudson River and Delaware River at spawning time. Surprisingly, we found that only 84% of river-of-origin genetic assignments corresponded with the spawning river in which the fish were detected by the acoustic arrays.