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Fungi and bacteria associated with bark beetles can facilitate successful tree colonization, and, in some cases, these fungi act as pathogens of trees. The red turpentine beetle (RTB, Dendroctonus valens) is a bark beetle native to North America that colonizes stressed pines, rarely killing healthy trees. The fungal communities associated with RTB adults, larval galleries, and control tree phloem from red pine (Pinus resinosa) and white pine (P. strobus) forests in the Great Lakes region of the United States were characterized using both culture-independent and culture-dependent methods. Similarly, the bacterial communities associated with RTB adults in the same region were characterized using a culture-independent method. There were significant differences between the adult beetle fungal communities and the tree-based fungal communities. Culture-independent sequencing of RTB adults showed high abundances of the fungal order Filobasidiales (red pine: 28.71% relative abundance, white pine: 6.91% relative abundance), as well as the bacterial orders Enterobacterales (red pine: 53.72%, white pine: 22.15%) and Pseudomonadales (red pine: 15.86%, white pine: 12.91%). In contrast, we isolated high amounts of fungi in the orders Pleosporales (red pine: 21.79%, white pine: 15.90%) and Eurotiales (red pine: 15.38%, white pine: 16.51%) from the adult beetles by culturing. Culture-independent sequencing of beetle galleries yielded high abundances of fungi in the orders Helotiales (red pine: 22.23%, white pine: 23.21%), whereas culture-based isolation from the same galleries yielded high amounts of Eurotiales (red pine: 17.91%, white pine: 17.91%), Hypocreales (red pine: 16.42%, white pine: 16.42%), and Ophiostomatales (red pine: 23.39%, white pine: 23.39%). This contrasts with the culture-independent method, where, likely due to limitations in the sequencing method, the Ophiostomatales accounted for only around 2% of the fungi from RTB galleries in both pine species. We observed a high species-level diversity of Ophiostomatales associated with RTB, isolating 14 species from the Great Lakes region. Leptographium terebrantis, a species that has been described in association with RTB throughout the United States, was the most common species (e.g., >35% of the Ophiostomatales relative abundance in red pine environments and >14% of the Ophiostomatales relative abundance in the white pine environment). This study enhances our understanding of RTB-associated fungi and bacteria in the beetle’s native range at both the community and species levels.