The species Syagrus romanzoffiana is a monocot belonging to the family Arecaceae; it is a palm endemic to South America and is widely distributed throughout the southeast and south of Brazil, including the State of Rio Grande do Sul. To estimate the genetic diversity and structure of the species in the watersheds of Rio Grande do Sul, five simple sequence repeat markers were used to analyze 90 individuals, representing three populations. The genetic structure of S. romanzoffiana groups was evaluated through the analysis of molecular variance and a multivariate analysis based on Nei’s genetic distance. The evaluated populations showed significant intrapopulation molecular variation (F_ST = 0.11). The observed heterozygosity (H_O = 0.17) was lower than the expected heterozygosity (H_E = 0.75). These data indicate that although the number of individuals is high, the diversity is low for some indices. Our findings suggest that further studies are needed, particularly on the genetics of natural populations of S. romanzoffiana in Rio Grande do Sul State, to fully characterize their genetic diversity and structure and determine strategies and priority areas for species conservation.