摘要： Background: Aspergillus flavus is a major cause of severe non‐invasive fungal infections in the Middle Eastern countries. However, it is difficult to distinguish A flavus from A oryzae. Objectives: To assess the potential of matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOF MS) in discriminating between A flavus and A oryzae and compare it with β‐tubulin gene sequencing. Methods: We used the Bruker Daltonik MALDI‐TOF MS system to analyse 200 clinical and environmental A flavus isolates and one A pseudonomius and one A alliaceus (Aspergillus section Flavi) isolate a priori identified as such by sequencing of the β‐tubulin gene. Results: All 200 A flavus isolates were identified at the genus level and 176 (88%) at the species levels by MALDI‐TOF MS based on the spectral log‐scores (≥2.0 and 1.7‐1.99, respectively); among them, only 18 (10.2%) were confirmed as A flavus, whereas 35 (19.9%) were identified as A oryzae and 123 (69.9%) as A flavus/A oryzae. Aspergillus pseudonomius and A alliaceus were misidentified as A flavus and A parasiticus with log‐score values of 1.39 and 1.09, respectively. Conclusions: The results indicate that the commercially available Bruker Daltonik MALDI‐TOF MS score database cannot separate A flavus and A oryzae species. We also showed that establishment of an in‐house library is a useful tool to discriminate closely related Aspergillus species, including A flavus and A oryzae.