Members of the recently introduced fungal genus Rasamsonia (formerly included in the Geosmithia genus) have been described as emerging pathogens in immunosuppressed hosts or patients with cystic fibrosis (CF). Rasamsonia species have often been misidentified as Penicillium or Paecilomyces because of similar morphological characteristics. We validated a commercially available real-time PCR assay (Primerdesign™, UK) for accurate detection of species from the Rasamsonia argillacea complex. First, we tested this assay with a collection of 74 reference strains and clinical isolates and then compared the PCR with cultures of 234 respiratory samples from 152 patients with CF from two University Hospitals in Germany and France. The assay reliably detected the three main species within the Rasamsonia argillacea species complex (R. argillacea, R. piperina, R. aegroticola), which are typically encountered in CF patients. The limit of DNA detection was between 0.01 and 1 pg/μL. Analysis of the DNA extracts from respiratory specimens of CF patients revealed that four out of the 153 patients studied (2.6%) were colonized with R. argillacea species complex. Two species from the R. argillacea complex grew in the parallel cultures from the same patients. In one patient the PCR was positive 5 months before culture. The real-time PCR assay is a sensitive and specific method for detecting the three most important species of the R. argillacea species complex encountered in the CF context. Detection of these emerging pathogens in respiratory secretions from CF patients by this novel assay may increase our understanding of the occurrence and epidemiology of the R. argillacea species complex.