Hancornia speciosa Gomes, popularly known as mangabeira, is a fruit tree belonging to the Apocynaceae family, native to the Brazilian Cerrado. The fruit is widely used by the local population as an alternative source of income. Limited information is available about this species, which increases the difficulty of conserving its genetic resources and exploiting mangabeira as an economic resource. The objective of this research was to evaluate the genetic diversity and genetic structure of H. speciosa from Chapada dos Guimarães. Twenty-four trees and ten inter simple sequence repeats (ISSR) primers were evaluated. Of the 57 bands obtained, 33 (57.89%) presented polymorphism. The analysis using Structure defined two different clusters (K = 2), which were consistent with the unweighted pair group method with arithmetic mean (UPGMA) clustering analysis. The number of observed alleles (Na = 1.58), number of effective alleles (Ne = 1.29), Nei’s genetic distance (He = 0.18) and Shannon index (I = 0.27) were considered low among the population. The analysis of molecular variance (AMOVA) revealed that the variability within the cluster (83.39%) was higher than among clusters (16.61%). ISSR primers proved effective for detection of genetic polymorphism in H. speciosa, and could be utilized for strategies that aim at conservation, plant breeding programs, and commercial use.