Mitochondrial DNA sequence variation in the 16S rRNA gene was used to estimate the phylogenetic relationships of Austropotamobius pallipes. The program ModelTest was used to test alternative models of evolution for our data using likelihood ratio tests. Both the minimum evolution tree with the HKY85 model and the maximum likelihood analysis supports the separation of two major clades (A and B) and three clades within clade A. The two major groups A and B showed genetic differentiation of 4.6% and could be in accordance with the classification on the specific status of A. italicus and A. pallipes. Within clade A, three clades were found corresponding to crayfish sampled in Spain-Italy-France, Austria, and Slovenia. In accordance with morphological data extracted from recent papers, a new classification based on the presence of three subspecies (italicus, carinthiacus, and carsicus) within A. italicus is proposed.