A thorough taxonomic knowledge about putative animal reservoirs of transmissible diseases is an absolute prerequisite to any ecological investigation and epidemiological survey of zoonoses. Indeed, accurate identification of these reservoirs is essential for predicting species-specific population outbreaks and therefore to develop accurate ecological control strategies. The systematic status of sand rats (genus Psammomys) remains unclear despite the pivotal role of these rodents in the epidemiology of Zoonotic Cutaneous Leishmaniasis (ZCL) disease as sand rats are the main known reservoir hosts of the protozoan parasite Leishmania major. In the present work, we expose morphological, biochemical, genetic and cytogenetic evidence supporting the identification of at least two cryptic species within the genus Psammomys in Tunisia. First, significant morphometric differences were observed and were correlated associated with external features and biogeographic origins. Second, differences in patterns of two isoenzymic systems (Glutamate Oxaloacetate Transaminase (GOT) and 6-PhosphoGluconate Dehydrogenase (6PGD)) were found, which makes it possible to amount these isoenzyme characters to two diagnostic loci. Third, based on the mitochondrial cytochrome b (cyt b) gene, a high magnitude of genetic distance (13.89%) was also observed. Fourth, cytogenetic analysis showed that these two populations groups differ in their diploid chromosome numbers, i.e. 2N=46 versus 2N=48. We consider that all these variations are enough important to be considered as demonstrative and we propose that these two lineages should be considered as two distinct species that we refer to the fat sand rat Psammomys obesus Cretzschmar, 1828 and the thin sand rat Psammomys vexillaris Thomas, 1925. Implications of such results on the eco-epidemiology of ZCL in Tunisia are discussed.