Dalbergia nigra is a long-living tree endemic to the Atlantic Rain Forest. Due to its high commercial value, this species has been widely exploited for timber production and is now endangered. It is widely known that understanding patterns of genetic structure is paramount for conserving threatened species. We analyzed the genetic diversity of 140 individuals from four different forest fragments in the southern region of Bahia, Brazil, to verify the possible effects of fragmentation on these populations and provide information for conservation initiatives. High polymorphism levels were detected from the genotyping of nine microsatellite loci (mean HE = 0.733). All populations showed high genetic diversity; however, a reduction of genetic diversity was detected in each population (HO < HE). The average fixation index was high and significant (f = 0.167), which could be due to the occurrence of inbreeding, the Wahlund effect, reproductive system, or from null alleles. Genetic differentiation among populations was high (mean θP = 0.118), suggesting strong isolation, a pattern consistent with historically low gene flow. The Bayesian analysis revealed five different genetic groups, among which three groups correspond to three different forest fragments, and two groups showed the genetic subdivision of individuals from the other forest fragment. Based on our results, the suggested conservation strategy for D. nigra populations in the southern region of Bahia, Brazil, involves high environmental investments to protect all sampled forest fragments and individuals. Another strategy would be to collect seeds from all individuals from the sampled fragments and start a new population with human interference in its evolutive history inside a protection unit.