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Ectomycorrhizal (EM) fungi form symbiotic associations with plant roots that regulate nutrient exchange between forest plants and soil. Environmental metagenomics approaches that employ next-generation sequencing show great promise for studying EM symbioses; however, metatranscriptomic studies have been constrained by the inherent difficulties associated with isolation and sequencing of RNA from mycorrhizae. Here we apply an optimized method for combined DNA/RNA extraction using field-collected EM fungal–pine root clusters, together with protocols for taxonomic identification of expressed ribosomal RNA, and inference of EM function based on plant and fungal metatranscriptomics. We used transcribed portions of ribosomal RNA genes to identify several transcriptionally dominant fungal taxa associated with loblolly pine includingAmphinema,RussulaandPilodermaspp. One taxon,Piloderma croceum, has a publically available genome that allowed us to identify patterns of gene content and transcript abundance. Over 1500 abundantly expressedPilodermagenes were detected from mycorrhizal roots, including genes for protein metabolism, cell signalling, electron transport, terpene synthesis and other extracellular activities. In contrast,Pilodermagene encoding an ammonia transporter showed highest transcript abundance in soil samples. Our methodology highlights the potential of metatranscriptomics to identify genes associated with symbiosis and ecosystem function using field-collected samples.