mtDNA-RFLP, a technique commonly used for molecular identification of species, was used to distinguish between morphologically similar crayfish species Austropotamobius torrentium and Astacus leptodactylus in this study. The mitochondrial COI gene was amplified and digested using 15 restriction endonucleases to establish a restriction map. The COI gene sequence contained recognition sites for three of the restriction endonucleases (SspI, KpnI, and RsaI) and produced two fragments and three genotypes for Austropotamobius torrentium. However, the Astacus leptodactylus COI gene did not have restriction sites for these three enzymes. Thus, a mtDNA-RFLP using these three endonucleases can be used to successfully distinguish between these two species of crayfish.