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.To assess the potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in discriminating between A. flavus and A. oryzae and compare it with β-tubulin gene sequencing.We used the Bruker Daltonik MALDI-TOF MS system to analyze 200 clinical and environmental A. flavus isolates and one A. pseudonomius and one A. alliaceus (Aspergillus section Flavi) isolates a priori identified as such by sequencing of the β-tubulin gene.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.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.