The sturgeons (order Acipenseriformes) are caviar producers and some of the most valuable fish species worldwide. Due to different reasons, wild populations are now at the brink of extinction. The high demand for caviar has led to the development of aquaculture for restocking and caviar production. Since the caviar from different species has different prices depending on the quality and attempts of commercial fraud based on species substitution have been found, correct species identification is more than necessary. We report a new multistep methodology for an accurate species identification based on both nuclear and mitochondrial markers. Our test integrates data from the analysis of microsatellites (Afu19, Afu34, Afu39, Afu54, Aox27, AoxD234, AnacC11 and AnacE4), nuclear gene markers (RPS7, vimentin and rhodopsin) and mtDNA barcoding to give a reliable molecular diagnostic for five sturgeon species (Huso huso, Acipenser stellatus, Acipenser ruthenus, Acipenser gueldenstaedtii and Acipenser baerii). In addition to species identification, our methodology allows the identification of bester, sterbe and best beluga hybrids, but also the identification of hybrids of unknown origin. This methodology has a good potential to contribute to the conservation of highly threatened sturgeon populations and also to the traceability of their products.