Abstract:[Background] The morphology and genome between Aspergillus flavus and A. oryzae were highly similar, which made it very difficult to distinguish with each other. [Objective] The aim of this study was to establish an accurate method for distinguishing A. flavus with A. oryzae. [Methods] Twenty-two standard strains were used to study the five traditional identification methods of A. flavus and A. oryzae, including the traditional morphological identification method, enzyme linked immunosorbent assay (ELISA), the toxin-secreting culture medium method, the phylogenetic tree method and the detection method for toxin genes. [Results] There are differences or complements among the results of different methods. [Conclusion] A single method used to distinguish the two species would have a potential risk, while a polyphasic approach could resolve this problem.