In conclusion, the results provided in this work show that: 1—the transferability of microsatellite markers is not an efficient strategy on A. flaccida; 2—the ISSR markers are a powerful tool to genetic studies on this species; 3—the populations of A. flaccida of grassy vegetation of southern Atlantic Forest have high genetic variability and are structured; 4—human activities (deforestation and folk medicine use) seems to influence the patterns of genetic variability in the natural populations of the species. The lack of previous genetic data of A. flaccida does not allow drawing any inferences about genetic drift on the species, but our results give support to the conclusion that the species is not at risk of extinction in the short term in the studied regions.