Abstract: Genetic variability and divergence among natural populations of Jaculus jaculus and J. orientalis in Tunisia were examined by electrophoretic analysis of 16 enzymatic proteins encoded by 23 genetic loci. Low levels of genetic variability were found among populations of both species in comparison to those of other rodent and mammal species of which data are available. In J. jaculus populations, the mean level of observed heterozygosity (H_obs) ranged from 0.08 to 0.19, while the mean percentage of polymorphic loci (P) ranged from 26.2% to 45.2% and the mean number of alleles per locus (A) ranged from 1.1 to 1.4. Nevertheless, the mean values were 0.10 to 0.15, 29.3% to 44.1% and 1.1 to 1.7, respectively, for J. orientalis. In addition, populations of the two species have revealed a lower degree of genetic differentiation (F_st=0.0017 for J. jaculus and 0.0019 for J. orientalis). Moreover, F_st was 0.607, P<0.05 between populations of the two species, indicating that they were highly genetically diverged from each other. The present data assures the previous results on the validity of the present taxonomic situation of the two species and emphasis on the effect of geographic factors (environments type and bioclimatic stages) on the genetic structure of both species.