Hymenaea courbaril L. is a tropical tree species with economic and medicinal potential largely used in programs for the recovery of degraded areas. The use of molecular markers in genetic studies can help in the definition of forest restoration strategies. This study was conducted to analyze the genetic diversity among H. courbaril (‘jatobá’) genotypes naturally occurring in the north of Mato Grosso State, Brazil, using ISSR markers, aiming to contribute to the development of seed collection and seedling production strategies. Twenty-four H. courbaril individuals were sampled by collecting leaves for DNA extraction by the CTAB method. The individuals were genotyped using 14 ISSR primers, resulting in the amplification of 87 bands in total, 65 of which were polymorphic. The ISSR markers presented average polymorphism information content of 0.44. Dissimilarity values based on the Jaccard coefficient ranged from 0.12 to 0.52. The most divergent individuals were 3 and 22, whereas 14 and 19 were the most genetically similar. The UPGMA and Tocher clustering methods indicated the formation of four distinct groups, revealing genetic variability among the 24 H. courbaril genotypes. Therefore, all the evaluated genotypes can be used for seed collection and seedling production, but the most divergent individuals should be prioritized.