Ambrosia beetles from the scolytine tribe Xyleborini (Curculionidae) are important to the decomposition of woody plant material on every continent except Antarctica. These insects farm fungi on the walls of tunnels they build inside recently dead trees and rely on the fungi for nutrition during all stages of their lives. Such ambrosia fungi rely on the beetles to provide appropriate substrates and environmental conditions for growth. A small minority of xyleborine ambrosia beetle-fungal partnerships cause significant damage to healthy trees. The xyleborine beetle vectors a (Hypocreales) fungus that is lethal to balsa ( (Malvaceae)) trees in Ecuador. Although this pathogenic fungus and its associated beetle are not known to be established in the United States, several other non-native ambrosia beetle species are vectors of destructive plant diseases in this country. This fact and the acceleration of trade between South America and the United States demonstrate the importance of understanding fungal plant pathogens before they escape their native ranges. Here we identify the fungi accompanying ambrosia beetles collected in Ecuador. Classification based ribosomal internal transcribed spacer 1 (ITS) sequences revealed the most prevalent fungi associated with are sp. and sp. (Microascales: Microascaceae), which have been confirmed as ambrosia fungi for xyleborine ambrosia beetles, and sp. (Hypocreales), which is a diverse genus found abundantly in soils and associated with plants. Phylogenetic analyses of the strains based on ITS, translation elongation factor (), and two subunits of the DNA-directed RNA polymerase II ( and ) identified them as sp. AF-9 in the Ambrosia Clade (AFC). This species was previously associated with a few xyleborine ambrosia beetles, most notably the species complex (Eichhoff 1868) (Curculionidae: Scolytinae: Xyleborini). Examination of ITS and sequences showed a close affinity between the isolated from spp. and other xyleborine-associated as well as the soil fungus . This characterization of ambrosia fungi through DNA sequencing confirms the identity of a putative plant pathogen spread by beetles and expands the documented range of and ambrosia fungi.