Three species of scorpions have been reported from Tunisia: Androctonus amoreuxi Audoin, A. aeneas Koch, and A. australis L. The latest has been divided in two subspecies: A. australis garzonii Goyffon & Lamy and A. australis hector Koch. Despite the public health importance of these animals, which cause several cases of poisoning each year, nothing is know about the genetic diversity of the group. To gain a better understanding of the group, we studied the variation of rDNA sequences of the ITS1, 5.8S, and ITS2 region of 14 representatives of the four taxa in Tunisia. The main result is the high polymorphism of the ITS regions. In some instances in both intra- and interspecific comparisons, it was difficult to unambiguously align the sequences. However, some representatives of different species were relatively similar, so that it appeared difficult to recognize the species on the basis of these sequences. A phylogenetic analysis was conducted that inquires the validity of the subspecies status of A. australis garzonii and A. australis hector. Moreover, the taxonomic status of A. aeneas is also questioned. Our studies highlighted the need of a revision of the taxonomy of the scorpions in Tunisia; however, the use of other genetic markers will be necessary to solve this question.