Fungi form a ubiquitous group of largely inconspicuous organisms that play key ecological roles in terrestrial nutrient cycling. The typically subterranean or otherwise unnoticeable nature of fungal life has left mycology with a partial understanding of fungal biology, and progress is hampered by factors such as the difficulty to delimit species and individuals of fungi and the sparsity of discriminatory morphological and physiological characters for reliable species identification. These and other complications have paved the way for DNA sequence data as an important source of information in mycology, and there are now twenty years’ worth of fungal sequence data available for scientific purposes. However, issues of data reliability and generality impede the use of publicly available fungal DNA sequences. The UNITE database for molecular identification of fungi (http://unite.ut.ee) was built as a response to the difficulties facing anyone seeking to identify environmental samples of fungi to species level using molecular data and the major international sequence databases. The present study describes the UNITE database and examines the role of UNITE in the light of emerging sequencing technologies, notably massively parallel (“454”) pyrosequencing. Environmental sampling of fungi is discussed from a taxonomic perspective.