Polyploidization has played an important role in vertebrate evolution. Acipenseridae bring clear examples of polyploidy ancestry and, also, polyploidization seems to be an ongoing process in these fishes. In the present study, the genetic origin of six triploid specimens morphologically determined as Acipenser ruthenus from commercial aquaculture was analyzed using a combination of mitochondrial and nuclear markers. A further five successive statistical analyses including median joining of mitochondrial DNA control region sequences, principal coordinate analysis (PCA), factorial correspondence analysis (FCA), STRUCTURE assignation, and NewHybrids status determination for microsatellite data were applied for the clarification of the origin of one extra chromosome set added in these triploids genomes. Although interspecific hybridization had been suggested as a source of these triploids, the statistical analyses showed that the investigated triploids originate from autotriploidization rather than from interspecific hybridization. Therefore, we conclude that a combination of molecular markers with suitable statistical analyses should be used to verify the origin of unusual ploidy level. Evidently, such an approach is critically essential in aquaculture, where interspecific hybridization is very common and usually detected by changes in ploidy levels only.