Summary• Here, the reliability of published fungal nucleic acid sequences is tested by the critical re-evaluation of 206 named sequences obtained from public-access databases.• Sequences from the ribosomal RNA (rRNA) gene cluster were examined as these are commonly used to establish fungal phylogeny and evolution, and are also increasingly employed in the identification of fungi from nonculture based studies.• Fifty-one rRNA internal transcribed spacer (ITS) sequences were obtained for species of Amanita , 55 ITS sequences were obtained for species of Phoma and 100 rRNA small subunit sequences were obtained from representative genera of the order Helotiales. In each case, the fungal group was selected partly on the basis of sequences deposited by three or more laboratories in order to avoid sample bias. The results suggest that up to 20% of the sequences available for each group may be unreliable, and this proportion is supported by additional informal observations.

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