Aiming to contribute to the reduction of the scientific gap regarding the genetic data of A. flaccida, this work sought to answer the following questions: 1—The transferability of microsatellite markers is an efficient strategy for obtaining genetic data from A. flaccida?; 2—ISSR markers are efficient for obtaining population genetic data in A. flaccida?; 3—The populations of A. flaccida of the Atlantic Forest are structured? and 4—Can high utilization of this species in folk medicine to influence the patterns of genetic variability in the natural populations. The answers to these questions will help determine the real state of A. flaccida conservation in the grassy ecosystems of southern Brazil and, if necessary, contribute to the elaboration of conservation strategies and rational exploitation of the species. Additionally, the data obtained here may help us understand the patterns of maintenance and expansion of the plant populations of the grassy ecosystems in the Atlantic Forest of southern Brazil from a historical perspective.