Genetic evaluations in breeding programs of critically endangered species are essential not only to characterize and maintain genetic diversity, but also to improve both effectiveness and monitoring of repopulation. The European sturgeon was the most widespread sturgeon species all over Europe, but its abundance has been drastically reduced to be now on the brink of extinction. Only a single population is surviving in the Gironde-Garonne-Dordogne watersheds (France) where the last known natural spawning occurred in 1994. In the years that followed, a sturgeon restoration program has been launched to build up a broodstock and repopulate this watershed. In the present study, a microsatellite multiplex assay was developed to characterize genetically the relict population actually reduced to 70 captive-wild spawners and the seven generations (2007-2014) issued from the breeding program. Our genetic assay proved highly reliable and powerful in relatedness analysis and assignment approaches, and is thus promising to help the conservation-restoration actions of the species. Mains aims include assessing the genetic variability of broodstocks and F1 juveniles, estimating relatedness and effective population size within captive individuals, providing molecular tags for released individuals, and assisting in the evaluation of survival of stocked individuals according to the parents used and the stocking practice. Here, we will present the obtained data and their usefulness for discussing suitable strategies for the genetic management and conservation of this almost extinct species.