The cytochrome oxidase subunit I (COI) gene was amplified and analyzed for 70 Mediterranean Chondrosia reniformis, collected from eight localities in Tunisia. Polymorphism results revealed high values of haplotype diversity (Hd) and very low nucleotide diversity (π). Thus, these results suggest that our sponge populations of C. reniformis may have undergone a bottleneck followed by rapid demographic expansion. This suggestion is strongly confirmed by the results of neutrality tests and “mismatch distribution”. The important number of haplotypes between localities and the high genetic differentiation (Fst ranged from 0.590 to 0.788) of the current C. reniformis populations could be maintained by the limited gene flow Nm (0.10 - 0.18). Both haplotype Network and the biogeographic analysis showed a structured distribution according to the geographic origin. C. reniformis populations are subdivided into two major clades: Western and Eastern Mediterranean. This pattern seems to be associated with the well-known discontinuous biogeographic area: the Siculo-Tunisian Strait, which separates two water bodies circulating with different hydrological, physical, and chemical characteristics. The short dispersal of pelagic larvae of C. reniformis and the marine bio-geographic barrier created high differentiation among populations. Additionally, it is noteworthy to mention that the “Mahres / Kerkennah” group diverged from Eastern groups in a single sub-clade. This result was expected, the region Mahres / Kerkennah, presented a particular marine environment.