Hesperozygis ringensis a native and endemic species of Rio Grande do Sul. This shrubby plant belonging to the Lamiaceaeis found in restricted locations in the sandy and stony soils of some municipalities. Hesperozygis ringensis known as "flea beater" due to the antiparasitic properties of the chemical compounds in its essential oil. However, studies focusing on populations monitoring and their genetic characterization are insufficient. The objective of this work was to verify the genetic diversity of H. ringenspopulations using populations from four municipalities. The leaves were collected and after stored and transported in silica gel. Total DNA was extracted using the CTAB method, and amplifications were performed with seven ISSR primers. The data were analyzed with GenAlEx, Structure and Power marker software. The amplifications resulted in 384 loci and the percentage of polymorphic loci averaged 59.44%. Low genetic diversity indices were recorded, with average Shannon index (I) = 0.267 and diversity (h) = 0.170. The Mantel test indicated that there is no significant correlation between genetic distance and geographical distance. The AMOVA showed that there is more variance within populations (89%) than between populations (11%). The plots of PCoA and Bayesian inference showed that the species has good population structure. The new information contributed significantly to broadening the general understanding of the species. Strategies are needed in order to increase genetic diversity and to implement measures for the preservation of the habitats where the populations are located.