Drought and desertification are the major environmental constraints facing the Sahelian agro-ecosystems for decades. Assessing genetic diversity of native tree species is critical to assist ecosystems restoration efforts. Here we describe genetic diversity and structure of seven Balanites aegyptiaca L. natural populations distributed across the Sahelian-Saharan zone of Mauritania using 16 polymorphic ISSR primers. These generated 505 polymorphic bands. Polymorphism information content (PIC) varied from (0.13-0.29) with an average 0.23, marker index (MI) averaged 7.3 (range 3.3-10.3) and resolving power (RP) ranged from (4.53-14.6) with an average 9.9. The number of observed alleles (Na) ranged from (0.62-1.39), Effective number of alleles (Ne) varied from (1.26-1.37), Shannon's information index (I) ranged from (0.25-0.36). AMOVA analysis showed that 80% of the genetic variation was fined within populations, which is supported by a low level of genetic differentiation between population (GST = 0.21) and an overall estimate of gene flow among populations (Nm = 1.9). The dendrogram based on Jaccard's similarity coefficient and the structure analysis divided the seven populations into two main clusters in which two populations from the Saharan zone were grouped. Our results provide baseline data for genetic conservation programs of this Sahelian neglected crop and with an important econ-ecological role.