Dalbergia melanoxylon Guill. & Perr. is a highly valued species in the Ferlo region, and its wood is used for various purposes by local populations, including firewood, service wood, and timber. This multifunctionality has weakened its populations, and it has been identified as threatened in previous research. The aim of this study was to investigate the genetic variability of three populations (LC, VC, and YC) along a north–south rainfall gradient, to support the species’ sustainable management and conservation. Eighteen microsatellite markers were used, including eight nuclear (nSSR) and 10 chloroplast (cpSSR) markers, to test their transferability to D. melanoxylon and study the genetic variability. The results showed that all nSSRs and cpSSRs were transferable, however, only five nSSRs and one cpSSR were polymorphic and used in the downstream analysis. Based on the nSSRs, comparatively high, but non-significantly different, genetic variation was found in all populations (LC: He = 0.650, VC: He = 0.674, YC: He = 0.706), however, indications of inbreeding were detected. For the single polymorphic cpSSR, three haplotypes were identified. Haplotype diversity (Hd) varied in LC (Hd = 0.536), VC (Hd = 0.577), and YC (Hd = 0.674). Low to moderate genetic differentiation was observed between populations (Fst nSSR = 0.019 and Fst cpSSR = 0.133) suggesting recent fragmentation of populations that has not yet impacted genetic variation but could become more pronounced in future generations. This finding underscores the need for further research on effective management and conservation strategies for D. melanoxylon populations in the Ferlo region.