The consistent association of Paleotropical Dipterocarpaceae with ectomycorrhizal (ECM) fungi suggests that ECM status is an ancestral character in the family. Despite its distinctive morphology, Pakaraimaea dipterocarpacea, a Neotropical Dipterocarpaceae endemic to the Guayana Region, is phylogenetically related to the Paleotropical Dipterocarpaceae. The confirmation of P. dipterocarpacea ECM status would indicate that Paleotropical Dipterocarpaceae and P. dipterocarpacea probably had a common ECM ancestor. Mycorrhizal colonization of P. dipterocarpacea was assessed, and ECMs were recorded using histological and molecular methods. P. dipterocarpacea was highly colonized by typical ECMs, and several ECM fungal taxa belonging to Clavulinaceae, Sebacinaceae, Cortinariaceae and Amanitaceae were identified. This paper provides the first documented evidence of ECM in a neotropical genus of Dipterocarpaceae and indicates that ECMs possibly evolved in Gondwana in ancestors of Dipterocarpaceae before the separation of South America from Africa by the Atlantic, c. 135 million years ago. The observation of Sebacinaceae and Clavulinaceae suggests that broad host range fungi are important components of P. dipterocarpacea ECM communities.