Brazil nut is a native Amazon species of high commercial value classified as vulnerable in terms of extinction risk due to marked illegal-burning activity and agricultural-frontier expansion processes occurring in the region. This study was undertaken to analyze the genetic diversity within and between two natural Brazil-nut populations occurring spontaneously in the Southern Amazon region spaced 50 km apart, both of which were located in the municipality of Alta Floresta, northern Mato Grosso state, Brazil. These are rural areas and samples were from native forest patches. Leaf samples were collected from 86 plants from distinct areas; 36 were from population AGRO (Agrocondor II Farm, geographic coordinates 55º30' W and 9º00' S) and 50 from population CAR (Carolina Farm, geographic coordinates 57º00' W and 11º00' S). A molecular-diversity study was conducted using 11 microsatellite loci developed for the species. To determine the level of genetic diversity between and within subpopulations, we applied principal coordinate analysis, analysis of molecular variance, observed and expected heterozygosity, polymorphic information ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 18 (2): gmr18174 F.S. Vieira et al. 2 content (PIC), UPGMA-based clustering, and Bayesian inference structuring. Seventy alleles were found with the SSR markers, with an average PIC of 0.72. Average Ho and He were 0.43 and 0.82, respectively. AMOVA revealed that 81% of the variability is within populations, as found in other studies of Brazil-nut in the states of Pará, Acre and Amazonas. The dendrogram obtained by the UPGMA method and the clustering provided by Bayesian inference resulted in two and four groups formed, respectively. All 36 individuals of the AGRO population were allocated in Group I, and the 50 individuals of population CAR were allocated in Groups II, III, and IV. The two subpopulations have sufficient genetic variability for the composition of an in situ germplasm bank that can be used in breeding programs and in programs for the conservation of the species.