We assessed the ability of Fourier transform infrared spectroscopy (FT-IR) to differentiate three important and morphologically similar <i>Aspergillus</i> species: <i>A. ochraceus</i> and <i>A. westerdijkiae</i>, and <i>A. niger</i>. Fungi were processed by two methods, powdered mycelia and conidiospore-saline solution, and then recorded in a spectrometer. Second derivatives with nine points of smoothing were applied as spectra data pretreatment. Partial least squares regression was used for the species comparison models and a prediction test was used to evaluate the models. The powdered-mycelia methodology correctly identified 100% of the prediction test set to discriminate <i>A. niger</i> from <i>A. ochraceus</i> and <i>A. westerdijkiae</i>; in addition, it had a 86.6% success rate in discriminating <i>A. ochraceus</i> and <i>A. westerdijkiae</i>. This is the first time a study assessed the ability of FT-IR to differentiate <i>A. niger</i>, <i>A. ochraceus</i>, and <i>A. westerdijkiae</i>, and we believe this technique is very promising for classifying and distinguish fungi isolates.

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