Baobab (Adansonia digitata L) belonging to Bombacaceae family, is one of the most widely used indigenous priority tree species in sub-Saharan Africa, valued in the cosmetic industry for its seed oil, and powdery fruit pulp for juice making. Baobab has high potential for domestication in southern Africa, therefore understanding its genetic diversity and population structuring is warranted. The study investigated the level of genetic diversity and differentiation of five populations of A. digitata L. sampled from four diverse silvicultural zones in Malawi. Variation at nine microsatellite loci were examined in 150 individual trees. Low mean genetic diversity was expressed through genetic diversity indices: Nei’s genetic diversity (h, 0.18 ± 0.03), Shannon Information Index (I, 0.21 ± 0.07), observed number of alleles (na, 1.47 ± 0.10), effective number of alleles (na, 1.23 ± 0.04) and percentage polymorphic loci (pp, 48%). The low genetic variation found is attributed to the population growing in marginal areas of genetic centre of diversity of the species, anthropogenic factors and founder effects. Moderate genetic differentiation was observed among populations (Gst = 0.13) indicating the presence of a large number of common alleles resulting in a homogenisation effect. Clustering of individual trees by genetic similarity coefficients indicated that mainland trees were genetically closer than the trees on Likoma Island. Mantel’s test showed a weak positive insignificant correlation (Z = 0.12; P = 0.64) between genetic distance among populations and actual distance on the ground implying that geneflow was not directly influenced by isolation by distance. The results suggest that seed distribution and tree improvement should recognise the presence of ecotypes and conservation measures should protect all the populations due to existence of private alleles which are of adaptive importance.