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A better understanding of ectomycorrhizal symbiosis leads to numerous advancements in forest management and environmental protection. The morphological identification of the ectomycorrhizae often proves to be misleading. For this reason, in order to study the ectomycorrhizal fungi communities, a number of molecular methods that require the isolation of nucleic acids are being used. However, ectomycorrhizal root tips, low mass heterogenic material rich in inhibitors, are a recalcitrant substrate in DNA isolation. It is common for published studies to include some number of unidentified root tips in their results, in spite of diverse isolation protocols being available to researchers. This study aims to analyze the relationship between the collected fungal material and later isolation results, and to propose a DNA isolation protocol specifically optimized for ectomycorrhizal root tips. It was found that the taxonomic position can be used to predict the potential isolation efficiency, with Ascomycota being generally more difficult from which to isolate DNA. After a number of cell lysis and lysate purification methods were evaluated, the joined approach of mechanical and chemical lysis, followed by silica column purification, was found to provide the best results, even with recalcitrant material.