Hevea brasiliensis (Euphorbiaceae) is the primary species for tapped natural rubber and an economically important crop since the 1870’s. Plantation-grown trees of H. brasiliensis are susceptible to numerous pathogens, some which are resistant to traditional chemical control. Fungal endophytes obtained from the wild are often considered ideal candidates for developing biological controls for pathogens, however little knowledge of the fungal endophytes associated with wild rubber and related hosts exists. The objectives of this study were the following: 1) determine the composition of the fungal endophyte communities associated with wild H. brasiliensis and its close relatives in Micrandra using cultures and culture-independent methods; 2) determine the species identities of endophytes in the genus Diaporthe associated with the two host genera; and 3) assess the overall utility of automated and manual methods for curating operational taxonomic unit (OTU) characterization of endophytes. To accomplish these objectives, fungal isolates and DNA from 381 seedlings and 144 adults of Hevea and Micrandra from three Amazon Peru locations were characterized using sequences from the internal transcribed spacer regions (ITS) of the ribosomal DNA repeat unit for both cultures and culture-independent samples, the latter using metagenomics methods. To determine the species identities of the Diaporthe isolates, four loci were analyzed using phylogenetic methods. A comparison of the effects of manual and automated operational taxonomic unit (OTU) classification was performed by using four different methods. Trichoderma species were most frequently identified from adult trees while Diaporthe species were the most abundant from seedlings, suggesting host developmental stage may be a key determinant of tropical endophytic community assemblage. Twenty-one distinct Diaporthe lineages were recovered with seven described as new species. Manual OTU curation methods were less error-prone suggesting that algorithm adjustments are needed for currently used automated methods. This study resulted in a greater understanding of the diversity of endophytes, particularly Diaporthe spp., associated with wild rubber. Knowledge of fungal diversity, host life stage associations, and spatial distribution of Hevea and Micrandra endophytes will provide additional tools for integrated disease management approaches and aid development of successful biocontrol methods.