AbstractAimThe restoration of the extinct Adriatic population of Beluga sturgeon, an iconic species with economic and traditional relevance, is a priority in upcoming conservation strategies but it must not occur without deep prior knowledge on the current diversity distribution. We defined informed criteria for the reintroduction of Beluga in Italian rivers by analysing its contemporary and ancient diversity based on a multi‐markers approach.LocationPonto–Caspian and Adriatic basins.MethodsWe examined the distribution pattern of genetic diversity of the extant beluga populations by conducting genetic analysis on the mitochondrial dLoop and 27 nuclear microsatellites in 119 individuals from 3 geographical basins (Azov, Black and Caspian seas) and genomic analysis on 893 SNPs isolated through the 2bRAD approach in a subgroup of 92 samples. Mitochondrial information was also used to evaluate the variability of the extinct Adriatic population by analysing a few available museum samples.ResultsThe historical Adriatic sample cannot be traced back to any of the contemporary ones laying to hypothesize the presence of a past isolated population. Instead, mitochondrial data did not reveal any geographically based clustering possibly reflecting the deep paleogeographical changes experienced by those areas. The genomic approach allowed us to depict for the first time a clear and supported genetic differentiation between two areas (the Black–Azov and the Caspian basins) but microsatellites also revealed a signal of differentiation of the Azov sample, possibly related to historical management activities in that area.Main conclusionsThe outcomes of the study revealed an unprecedented amount of information that can provide great benefits to the establishment of ex situ Beluga broodstocks and support any future translocations. The provided guidelines should be taken as a reference for the upcoming restoring of the Italian extinct Beluga population and also for any future management activity of this species in its entire distribution range.