The hybrid sturgeon Acipenser baerii × A. schrenckii is the most widely cultured commercial sturgeon in China. However, its morphological similarity to the parental species frequently leads to misuse of germplasm in the breeding process, resulting in a decline in the quality of the sturgeon production. In this study, we have developed a protocol by using mitochondrial DNA barcoding and microsatellite locus analysis for the accurate identification of sturgeon species. Genetic distance and phylogenetic analysis based on the mitochondrial COI segment showed that A. baerii exhibited the closest genetic relationship with orthogonal individuals A. baerii (♀) × A. schrenckii (♂). Conversely, A. schrenckii displayed the highest genetic similarity with reciprocal individuals A. schrenckii (♀) × A. baerii (♂). Additionally, genetic structure analysis and factor correlation analysis (FCA) were conducted using six microsatellite loci among 100 samples, including eight species and two hybrid sturgeon. The results showed that all samples, encompassing both hybrid sturgeon (A. baerii × A. schrenckii) and their parental species, were accurately grouped into ten clusters, thereby validating the precision of this species assignment method.