Eugenia uniflora L. (Myrtaceae) is a tree species widely distributed in South America suffering the effects of the exploitation of natural populations. In this study, we employed low coverage sequencing of the E. uniflora genome for mining of SSR markers. The de novo assembly generated 2,601 contigs with an average length of 1139 bp and spans 3.15 Mb. A total of 76 dimer, 33 trimer and two compound SSR loci were identified. Twelve selected SSR loci were employed to genotype 30 individuals from two natural populations. A total of 73 alleles were detected (mean A= 6.1) were observed, the mean effective number of alleles was Ae = 3.91, mean Ho was 0.23 and mean HE was 0.70). The mean Wright's within population fixation index was FIS = 0.66 and significant deviation of HWE was observed in all loci, except one. The FST between populations equaled 0.27. The levels of genetic diversity and structure estimated with these 12 SSR markers are in accordance with data from genetics studies performed on other tree species of the Pampa biome, presenting moderate to high polymorphism and may be employed in studies of species conservation measures and breeding programs.