Pooti (Hopea gregaria) is a medium size tree that can grow up to 35 m from the Dipterocarpaceae family. Since 1998, the International Union for Conservation of Nature (IUCN) has reported the pooti tree as endangered, and the species has a narrow distribution, found only in Southeast Sulawesi. One of the efforts to overcome the rarity of the Pooti plant is through a tree breeding program. However, this tree breeding program will only be successful with the support of genetic diversity information. The study of genetic diversity can be done using a molecular-based approach using Inter Simple Sequence Repeats (ISSR) markers. Samples of Pooti leaves used in the study were taken from Botanical Forest Park Nipa-Nipa (Tahura) and Protected Forest Nanga-Nanga, Kendari City, Southeast Sulawesi Province. A total of 50 samples were extracted using the CTAB (Cetyl Trimethyl Ammonium Bromide) method. Ten types of primers that have been carried out obtained seven primers that can produce bright polymorphic bands and can be continued for study genetic diversity, namely UBC 810, UBC 813, UBC 814, UBC 822, UBC 823, UBC 827, and UBC 830.