[Objective] Fungi are found everywhere and associated closely with human life. Identification of fungi is the basis of further scientific research and commercial application of fungal resources. In this study, 112 fungal strains preserved at the Agricultural Culture Collection of China (ACCC) were randomly chosen to test the application of rDNA internal transcribed spacer (ITS) sequences in their identification at the genus level. [Methods] rDNA ITS was sequenced from all the strains used in this study and compared with available sequences in GenBank using a BLASTn search for their generic identification. [Results] Among the strains used, 80 were consistent with their original generic determinations. Polymorphisms of DNA bases or impurity of sequence chromatograms reduced the similarity in comparison with the target sequence in GenBank. [Conclusion] rDNA ITS sequences can be used for confirmation of fungal strain identity at the level of genus. It is recommended that all the fungal strains prior to and in preservation in national microorganism collections should be checked through rDNA ITS sequence analysis and the name of strains should be treated with caution. The original determination and the history of used names of each strain should be kept in the database for further reference.