Cajui (Anacardium spp.) is a native fruit tree (small cashew) of the Brazilian Cerrado and possesses the potential for commercialization. However, cajui exploitation occurs exclusively through extractivism in the absence of conservation strategies. The lack of conservation strategies may lead to a decrease in genetic diversity of Anacardium. In this work, the genetic diversity and population structure of three natural populations in Sete Cidades National Park (PNSC; PI, Brazil) were assessed using ISSR analysis of 56 cajui accessions and two A. occidentale accessions (outgroup) from Pacajus (CE, Brazil). A total of 112 markers were obtained, 93 (83.04%) of which were polymorphic. The diversity indices of these populations indicated moderate levels of genetic diversity. According to AMOVA, 96.17% of the genetic variability lay within populations, with low genetic differentiation among populations (ΦST = 0.03828). Furthermore, STRUCTURE analysis indicated the existence of four connected genetic groups. The findings show that the individuals from the three collection sites did not represent different subpopulations, likely due to the high gene flow (Nm = 6.7) favored by the floral biology of Anacardium, pollinators and small-animal seed dispersers. This research identifies genetically divergent individuals (C-03, C-05, C-22, C-26, C-34 and C-39), which should be considered priority individuals for conservation and can inform conservation programs for Anacardium spp.