Background: Anaerobic fungi (Neocallimastigomycetes) are common inhabitants of the digestive tract of mammalian herbivores, where they play an important role in fibre degradation. The internal transcribed spacer 1 (ITS1) region is currently the molecular marker of choice for anaerobic fungal community analysis, despite its known size polymorphism and heterogeneity within pure cultures. The aim of this study was to assess the accurateness of anaerobic fungal ITS1 based barcoded amplicon sequencing for community composition analysis. The assessment was conducted using the following approach. Full-length ITS1 clone libraries from five pure cultures, representing the ITS1 region size range, were Sanger sequenced to generate a reference dataset. Barcoded amplicons of the same five pure cultures, and four different mock communities derived from them, were then sequenced using Illumina HiSeq. The resulting barcoded amplicon sequences were then assessed in relation to either the reference dataset (for the pure cultures) or the corresponding theoretical mock communities (derived from the reference dataset).Results: Annotation of sequences obtained from individual pure cultures was not always consistent at the clade or genus level, irrespective of whether clone libraries or barcoded amplicons were analysed. The minimum detection threshold of the barcoded amplicon method was above 1.5%, presumably due to the high number of PCR cycles used. Accuracy of two of the four mock communities was limited due to preferential amplification of smaller sized ITS1 regions. Of the 32 named anaerobic fungal clades, for which full ITS1 region sequence data is available, this preferential amplification is predicted to be an issue with only 6 of them (Orpinomyces 1a, Orpinomyces 1b, Orpinomyces 2, Cyllamyces 1, Cyllamyces 2 and Buwchfawromyces/SK2).Conclusions: Whilst anaerobic fungal ITS1 based barcoded amplicon sequencing is of value for environmental sample analysis, we conclude that the accuracy of the results is highly dependent on community composition due to preferential amplification of smaller sized ITS1 regions. Studies to date indicate that the D1/D2 region of the 28S rRNA gene is a promising alternative molecular marker. As such, there is an urgent need to obtain more reference sequences and develop an associated working taxonomic scheme in order to enable the suitability of the D1/D2 region as taxonomic marker for anaerobic fungal barcoded amplicon sequencing to be verified.