Dalbergia sissoo, a wind-dispersed tropical tree, is one of the most preferred timber tree species of South Asia. Genetic diversity and differentiation among natural populations of D. sissoo were examined for the first time. We found a relatively high level of genetic diversity in D. sissoo, both at the species level (percentage of polymorphic bands = 89.11%; H = 0.2730; I = 0.4180) and the population level (percentage of polymorphic bands = 68.7%; H = 0.239; I = 0.358), along with a relatively low degree of differentiation among populations (GST = 0.1311; AMOVA = 14.69%). Strong gene flow among populations was estimated, N m = 3.3125. The Mantel test suggested that genetic distances between populations were weakly correlated with geographic distances (R = 0.3702, P = 0.1236). The high level of genetic diversity, low degree of differentiation, strong gene flow, and weak correlation between genetic and geographic distances can be explained by its biological character and widespread planting. This information will be useful for the introduction, conservation and further studies of D. sissoo and related species.