Narrow-clawed crayfish (Astacus leptodactylus Eschscholtz, 1823 species-complex) is one of five European freshwater crayfish species. Even though widely distributed, it hasn’t been frequently studied and its taxonomy and systematics are unresolved. The results of a recent comparative morphometric character study revealed that morphometry of Asian and European populations differ significantly. In this research, for the first time, mitochondrial molecular markers (16S rRNA and COI) were used with the aim of elucidating the phylogenetic relationship between European and Asian populations of the narrow-clawed crayfish. Analyses included crayfish from Croatia, Bulgaria, Armenia, Russia, Poland and Turkey, and three different optimality criteria were applied. Phylogenetic relationships were reconstructed using the COI dataset, as well as the concatenated one (COI + 16S rRNA). For both datasets, congruent topologies were obtained and trees were characterised by the existence of two well supported phylogroups, one that included European populations, and the other Asian. Results indicate the presence of distinct evolutionary lineages within the A. leptodactylus species-complex, and corroborate previous results obtained using morphometric analyses.