Calotropis gigantea is well known for its aesthetic, medicinal, pharmacological, fodder, fuel, and fiber production potential. Unfortunately, this plant species is still undomesticated, and the genetic information available for crop improvement is limited. For this study, we sampled 21 natural populations of C. gigantea from two key areas of its natural distribution range (Bangladesh and China) and genotyped 379 individuals using nine nuclear microsatellite markers. Population genetic diversity was higher in Bangladesh than that observed in Chinese populations. Overall, a moderate level of genetic diversity was found (N-a = 3.73, H-E= 0.466), with most of the genetic variation detected within populations (65.49%) and substantial genetic differentiation (F-ST = 0.345) between the study regions. We observed a significant correlation between genetic and geographic distances (r = 0.287, P = 0.001). The Bayesian clustering, UPGMA tree, and PCoA analyses yielded three distinct genetic pools, but the number of migrants per generation was high (N-M = 0.52-2.78) among them. Our analyses also revealed that some populations may have experienced recent demographic bottlenecks. Our study provides a baseline for exploitation of the genetic resources of C. gigantea in domestication and breeding programs as well as some insights into the germplasm conservation of this valuable plant.