The ability to distinguish captive-bred and natural-origin individuals in the wild is critical for evaluating the impact of captive breeding programs on natural populations. Continued persistence of endangered pallid sturgeon (Scaphirhynchus albus) in the Missouri River is largely dependent on captive breeding efforts that spawn natural-origin adults in fish hatcheries and release their progeny into the wild. Prior to release, hatchery-origin individuals are physically marked so they can be distinguished from natural-origin individuals when recaptured. During the years 2004–2006, 24 unmarked juvenile pallid sturgeon tissue samples were collected in the Missouri River downstream of Gavins Point Dam, South Dakota, USA that were presumed natural-origin. However, these individuals were similar in size to hatchery-origin fish released in this area raising concerns that these individuals were actually hatchery-origin fish with lost or malfunctioning tags. We used microsatellite based parentage analysis to determine if the unmarked fish were members of hatchery families that had been released in this area. This retrospective genetic tagging approach revealed that 23 of 24 unmarked fish were indeed hatchery-origin. The origin of the remaining individual remains unknown because genetic samples were not available from all of the families released below the dam and the unassigned fish may have originated from one of these un-sampled families. These results provide important insight into the conservation status of endangered pallid sturgeon as well as provide data important for guiding management decisions. Our results also demonstrate the efficacy of using genetic tags as an alternative or complimentary approach to physically marking individuals.