The nuclear ribosomal DNA internal transcribed spacer (ITS) has been widely used to assess the fungal composition in different environments by deep sequencing. To evaluate the ITS in the analysis of fungal diversity, comparisons of the clustering and taxonomy generated by sequencing with different portions of the whole fragment were conducted in this study. For a total of 83,120 full-length ITS sequences obtained from the UNITE database, it was found that, on average, ITS1 varied more than ITS2 within the kingdom Fungi; this variation included length and GC content variations and polymorphisms, with some polymorphisms specific to particular fungal groups. The taxonomic accuracy for ITS was higher than that for ITS1 or ITS2. The commonly used operational taxonomic unit (OTU) for evaluating fungal diversity and richness assigned several species to a single OTU even with clustering at 99.00% sequence similarity. The clustering and taxonomic capacities did not differ between ITS1 and ITS2. However, the OTU commonality between ITS1 and ITS2 was very low. To test this observation further, 219,741 pyrosequencing reads, including 39,840 full-length ITS sequences, were obtained from 10 soil samples and were clustered into OTUs. The pyrosequencing results agreed with the results of the in silico analysis. ITS1 might overestimate the fungal diversity and richness. Analyses using ITS, ITS1 and ITS2 yielded several different taxa, and the taxonomic preferences for ITS and ITS2 were similar. The results demonstrated that ITS2 alone might be a more suitable marker for revealing the operational taxonomic richness and taxonomy specifics of fungal communities when the full-length ITS is not available.