Morphometric and karyological variations were analysed within the Moorish gecko Tarentola mauritanica sensu lato from Tunisia, and compared with already published data. The results show high levels of interspecific variability, indicative of the existence of two distinct lineages: the larger one T. fascicularis, widespread in the south, nearly identical to specimens from Libya and Egypt and the other one, T. m. mauritanica, confined to the northern and central regions, belonging to the common “European” and “North African” morphotype. The observed morphological variation appears to be related to microevolutionary events (phylogenetic variation) derived from a speciation event between the two taxa. We observed that these two lineages also differed in karyotype, such as the shape of some chromosomes and position of NORs. This study highlights the importance of Tunisia as a transition area between eastern and western lineages of Moorish gecko. However, additional genetic studies seem to be essential to infer evolutionary relationships among T. mauritanica populations' across North Africa.