Resumo. Jatobá (Hymenaea courbaril L.) is a species that shows ecological importance and occurs in different geographic regions in Brazil. Knowing and evaluating the existence of places with higher genetic variability can help in conservation programs and actions. In this context, the present study aimed to evaluate the genetic diversity and structure in natural populations of Jatobá with occurrence in Mato Grosso Amazon through ISSR markers. Fifty-four individuals were sampled in three populations, being one in the municipality of Marcelândia (MA=24) and two in the municipality of Alta Floresta (Comunidade Central AF=17 and Pista do Cabeça PC=13 individuals). Total genomic DNA was extracted from the leaf tissue by the CTAB method. The 54 individuals were genotyped with 10 ISSR primers. A total of 110 fragments were amplified, being 78.2% polymorphic. The greatest diversity indices were found in the population from MA (H=0.25; I=0.37 and %P=65.45). The genetic distance was higher among the populations of the two municipalities (0.17 between MA and AF; 0.16 between MA and PC). The analysis of molecular variance (AMOVA) indicated that 31.79 % of the total variance is among populations and 68.21 % within populations. There is genetic diversity in native populations of Jatobá in Mato Grosso Amazon. We recommend that individuals from both populations be preserved, in order to ensure the maintenance of genetic variability and the effective conservation of species in Mato Grosso Amazon.