The pre-symbiotic phase that precedes physical contact between symbionts is a crucial phase in determining their compatibility, allowing the formation of the ectomycorrhiza. A subtractive cDNA library representing the differentially expressed genes of the fungus Hydnangium sp. in the pre-symbiotic phase was constructed using fungal mycelia obtained through the in vitro mycorrhization technique. The fungus was cultured in the presence of Eucalyptus grandis roots, but with no contact between the hyphae and the root system of the host plant. Genes that code for proteins related to carbohydrate, amino acid, and energy metabolisms, transcription, and protein synthesis, cellular communication, signal transduction, stress response, transposons, and proteins related to the biogenesis of cell components were identified among the 131 expressed sequence tags. Expression of the genes that code for acetyl-CoA acetyltransferase, pyruvate dehydrogenase, ATP synthase, a voltage-dependent protein from the selective ion channel, and hydrophobin was evaluated by the RT-qPCR technique, confirming the activation of these genes in this phase of the association.