Mimosa tenuiflora (Willd.) Poir. (Fabaceae) has pharmaceutical potential and can be used to treat infections. It is also widely used as firewood and in the production of red ceramics, which makes the timber intensively harvested at the sites of its occurrence. In this study, we hypothesized that the M. tenuiflora populations in semi-arid vegetation present low genetic diversity owing to the intense historical exploitation of resources. We sampled 225 individuals distributed among 15 natural populations and performed several analyses. The DNA extracted was subjected to a Polymerase Chain Reaction using seven selected primers. A total of 70 fragments were obtained, and the percentage of polymorphic loci ranged from 58.57 to 81.43%. The genetic diversity of Nei and the Shannon index suggested low genetic diversity in the populations. The analysis of molecular variance indicated a higher proportion of genetic variability within the populations. There was no correlation between the genetic and geographical distances. The Bayesian analysis revealed that the populations are structured into four genetic groups. We detected genetic bottlenecks for 13 populations, including the most diverse ones. Although some populations showed evidence of a genetic bottleneck, none were significant for both models. Our results showed that M. tenuiflora is a threatened species and a priority for implementing conservation strategies. Moreover, genetic diversity results could be used for future breeding programs to meet the demand for the plant and expand wood commercialization without extracting this species from natural populations.