Objectives The aim of this study was: (i) to study the prevalence of triazole-resistant Aspergillus fumigatus isolates in the Netherlands; and (ii) to design rapid real-time PCR methods to identify such isolates. Methods A novel mixed-format real-time PCR assay is described for the detection of mutations leading to triazole resistance in A. fumigatus . One set of PCR primers and a probe carrying a single fluorescent label in combination with a double-stranded DNA fluorescent dye allow simultaneous detection of (a) specific mutation(s) as well as of the amplified product that serves as an internal amplification control. The method was applied to a random collection of 209 clinical isolates from throughout the Netherlands and was compared with phenotypic susceptibility testing. Results A total of four triazole-resistant isolates were identified, resulting in a prevalence of resistant isolates of <2%. All four isolates contained an identical combination of mutations leading to multi-triazole resistance, as reported before by others. Molecular testing results were 100% concordant with phenotypic susceptibility testing. Conclusions Although in specific patient populations the prevalence of resistance in A. fumigatus may be an emerging problem, in the general population it is still relatively low. The novel real-time PCR format allows rapid and reliable identification of such isolates.