1. The internal transcribed spacer (ITS) is used often in DNA metabarcoding of fungi. One disadvantage of its high variability may be a failure to classify operational taxonomic units (OTUs) when no similar reference sequence exists. We tested whether the 5.8S region, often sequenced with ITS2 but discarded before analysis, could provide OTU classifications when ITS2 fails. 2. We used in silico evaluation to compare classification success of 5.8S and ITS2 from the UNITE database when reference sequences of the same species, genus, or family were removed. We then developed an automated pipeline for a combined 5.8S–ITS2 analysis and applied it to mixed environmental samples containing many lineages that are underrepresented in databases. 3. ITS was clearly superior for species‐level classifications with a complete reference database, but 5.8S outperformed ITS at higher level classifications with an incomplete database. Our combined 5.8S‐ITS2 pipeline classified 3× more fungal OTUs compared to ITS2 alone, particularly within Chytridiomycota (27×) and Rozellomycota (6×). 4. Missing reference sequences led to the failure of ITS to classify many fungal OTUs at all, and to a significant underestimation of environmental fungal diversity. Using 5.8S to complement ITS classification will likely provide better estimates of diversity in lineages for which database coverage is poor.
