Dysoxylum binectariferum is an important timber and medicinal tree species distributed in the Western Ghats, India. The species is highly threatened by anthropogenic activities and has been extensively harvested in many parts of the Western Ghats. Populations of D. binectariferum are thus fragmented and reduced to incredibly small numbers. Here, we sampled six fragmented populations of D. binectariferum and used four polymorphic microsatellite loci to investigate population gene flow and genetic variation, as well as diversity in adults and seedlings. We found high levels of within-population diversity (mean alleles/locus = 4.875; mean HE = 0.63), low genetic differentiation between populations (FST = 0.16), and a consistent deficit of heterozygotes across all populations (mean FIS = 0.11). There was a significant reduction in allelic richness in seedlings (mean allele/locus = 3.29) compared to adults (mean allele/locus = 4.37), although both had similar levels of mean observed heterozygosity (Ho = 0.55). Decline in seedling allelic richness suggests the present populations have been affected by habitat fragmentation, and warrants consideration in conservation and management planning of D. binectariferum populations.