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The reintroduction of the extinct beluga sturgeon (Huso huso L.), an anadromous species with economic and traditional relevance, is a priority in next conservation strategies in Northern Italy. The EU-LIFE NATURA project aims to reintroduce the beluga sturgeon in the Po River basin through a captive breeding program. Critical requirements for the success of the program are river connectivity and knowledge of genetic diversity of the selected broodstocks to ensure self-sustainability of reintroduced populations. Here, the four broodstocks used for the reintroduction of beluga sturgeon have been genetically screened, genotyping 13 loci and sequencing mitochondrial DNA cytochrome b (Cyt b) gene and the entire mitochondrial DNA control region (D-Loop). The four broodstocks showed a medium-high level of nuclear genetic variability and the presence of two sub-populations, evidencing a total level of inbreeding coefficients able to sustain the good potential as future breeders. Mitochondrial analyses showed a genetic variability comparable to wild populations, further strengthening the positive potential of the investigated broodstock. Therefore, this study, showed how the degree of genetic diversity found within the four broodstocks used for H. huso reintroduction in the Po River basin could be suitable to ensure the success of the program, avoiding the inbreeding depression associated with founder effect and captive breeding.