Jerboas belonging to the genus Jaculus are widely distributed rodents inhabiting Palearctic desert and semi-desert areas. Previous studies on the lesser Egyptian jerboa Jaculus jaculus showed the existence of various morphological forms of controversial taxonomic status. They were sometimes related to two different species, J. jaculus and Jaculus deserti, although this has not been recognized in recent taxonomic updates. To clarify the systematic status of J. jaculus in Tunisia, we performed molecular (phylogenetic analyses of cytochrome b sequences), morphological (multivariate analyses of 13 skull measurements) and karyotypic (standard preparations from bone marrow cells) analyses on a number of specimens collected from ten localities. Our analyses revealed two monophyletic, well differentiated clades, with a mean genetic divergence value (K2P = 10.9 ± 0.01%), which is within the range of distances generally observed between rodent species. Morphometric analyses clearly separated populations of the two genetic clades from each other. However, karyotypes of individuals from both clades appeared similar. Individuals from both molecular clades/morphometric groups were found in sympatry in most of the localities sampled. These results, as obtained from a restricted area of the total distribution, suggest that there are two separate species within the currently accepted J. jaculus in Tunisia. Alternative hypotheses such as the occurrence of a strong, ancient phylogeographic structure, or the presence of pseudogenes, are also considered to account for the results obtained.