The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric – artificially joined – DNA sequences during PCR amplification or sequence assembly. Several software tools for chimera detection are available, but they rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. No such dataset is, however, freely available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for molecular identification of fungi. The dataset supports chimera checking throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was found to be well above 99.5%, and we used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is freely available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.