For more than 20 years, the cosmopolitan species Porcellionides pruinosus (Brandt, 1833) has been characterised by a controversial taxonomic status. This "species" has a high ecological plasticity, which enabled it to conquer different kinds of habitats. Besides that, this "species" exhibits a geographical variation in its morphological features and its reproduction pattern. In fact, some Tunisian populations had a seasonal reproductive period and while others showed a reproductive activity. A genetic analysis of rDNA 16S sequences has been performed to compare eight populations of P. pruinosus from different geographical sites, located in the north, centre, and south of Tunisia. Results reveal that the population of Tunis shows considerable variation in the primary structure of the sequences and has a substantial genetic diversity (F ST = 0.98 and F CT = 0.97). However, the genetic variation is low for the populations of Bella Regia and Bousalem (0.00041), and null between populations of Elfeija, Chebba, Monastir, Sbiba and Sned. Moreover, the genetic distance of the population of Tunis (≥ 0.35) compared to the other populations raises questions about the taxonomic status of this population. Also, in this latter population transitions are the most dominant variations, whereas for the other seven populations there are only two types of transversion substitutions. The results of the current study emphasise the controversial taxonomic status of Porcellionides pruinosus in Tunisia. Consequently, it would appear that the taxonomic status of P. pruinosus from Tunisia needs a re-evaluation.