Khaya senegalensis (African mahogany or dry-zone mahogany) is a high-value hardwood timber species with great potential for forest plantations in northern Australia. The species is distributed across the sub-Saharan belt from Senegal to Sudan and Uganda. Due to heavy exploitation and constraints on natural regeneration and sustainable planting, it is now classified as a vulnerable species. Here we describe the development of microsatellite markers for K. senegalensis using next generation sequencing in order to assess its intra-specific diversity across its natural range, which is a key for successful breeding programs and effective conservation management of the species. Next generation sequencing yielded 93 943 sequences with an average read length of 234 bp. The assembled sequences contained 1030 simple sequence repeats, with primers designed for 522 microsatellite loci. Twenty one microsatellite loci were tested with eleven showing reliable amplification and polymorphism in K. senegalensis. The eleven novel microsatellites, together with one previously published, were used to assess 73 accessions belonging to the Australian K. senegalensis domestication program, sampled from across the natural range of the species. STRUCTURE analysis shows two major clusters, one comprising mainly accessions from west Africa (Senegal to Benin) and the second based in the far eastern limits of the range in Sudan and Uganda. Higher levels of genetic diversity were found in material from western Africa. This suggests that new seed collections from this region may yield more diverse genotypes than those originating from Sudan and Uganda in eastern Africa.