Aim: Triazole resistance in Aspergillus fumigatus is associated with mutations in cyp51 genes, therefore, a single-tube multiplex PCR was proposed for rapid detection of such mutations. Methods: Relevant markers (n = 21) located in cyp51A and cyp51B were amplified in a multiplex reaction and subsequently analyzed by mini-sequencing. Results: A set of nonresistant A. fumigatus were tested. The markers F46, G89, M172, D255, L358, E427 and C454 located in cyp51A, as well as P394 and S35 from cyp51B, were found to be modified. Conclusions: A. fumigatus triazole resistance in Portugal is rare, nevertheless, some isolates showed alterations in the cyp51 genes. Multicenter studies with more isolates should better evaluate and validate the potential use of this method in clinical laboratories. The new methodology allows the addition of extra markers if described as relevant for A. fumigatus susceptibility to triazoles. First draft submitted: 27 March 2015; Accepted: 24 August 2015; Online: 30 October 2015