Abstract: In order to perform checks in the trade of fish species under the threat of extinction and with commercial importance such as sturgeon, the fish must be identified rapidly and precisely from any tissue and for this purpose morphometric and protein based determinations can be inadequate. In this study, Acipenser stellatus, Acipenser gueldenstaedtii and Huso huso sampled from Trabzon coasts were used. For RFLP and sequence analysis, the mitochondrial Cytochrome-b (mtDNA Cyt-b) gene was amplified by the help of PCR. From Cyt-b sequence data, it was observed that in the Maximum Parsimony (MP) dendrogram, 99% of the species were separated. Furthermore in the Cyt-b PCR product restriction enzyme analyses, it was determined that Hinf I and Rsa I enzymes were distinguishing for 3 species. In the multiplex PCR application in the mtDNA D-loop region, the product lengths were determined as 420 bp for A. gueldenstaedtii, 350 bp for H. huso and 260 bp for A. stellatus and they are distinctive for the species. The results showed that the multiplex PCR application is a method which is cheap and effective for the identification of sturgeon species in short duration.