The aim of this work was to document molecular epidemiology of Rasamsonia argillacea species complex isolates from cystic fibrosis (CF) patients. In this work, 116 isolates belonging to this species complex and collected from 26 CF patients and one patient with chronic granulomatous disease were characterized using PCR amplification assays of repetitive DNA sequences and electrophoretic separation of amplicons (rep-PCR). Data revealed a clustering consistent with molecular species identification. A single species was recovered from most patients. Rasamsonia aegroticola was the most common species, followed by R. argillacea sensu stricto and R. piperina, while R. eburnea was not identified. Of 29 genotypes, 7 were shared by distinct patients while 22 were patient specific. In each clinical sample, most isolates exhibited an identical genotype. Genotyping of isolates recovered from sequential samples from the same patient confirmed the capability of R. aegroticola and R. argillacea isolates to chronically colonize the airways. A unique genotype was recovered from two siblings during a 6-month period. In the other cases, a largely dominant genotype was detected. Present results which support the use of rep-PCR for both identification and genotyping for the R. argillacea species complex provide the first molecular evidence of chronic airway colonization by these fungi in CF patients.R asamsonia argillacea, which was first reported in clinical practice as a Penicillium species, then reclassified in the genus Geosmithia, and finally reassigned in 2013 to the new genus Rasamsonia, is now considered an emerging pathogen (1). Since its first report in humans as Penicillium emersonii (2), there has been an increasing number of cases reported in both dogs (3, 4, 5) and humans (6). Nevertheless, it is likely that the number of human infections caused by this fungus has long been underestimated because of the lack of specificity of its morphological features and to subsequent misidentifications with some Penicillium or Paecilomyces species.Only six species were previously recognized in the genus Rasamsonia described in 2012, i.e., R. argillacea, R. composticola, R. brevistipitata, R. byssochlamydoides, R. cylindrospora, and R. emersonii (7). However, multilocus sequence analysis of several R. argillacea isolates revealed a clustering which superimposed some phenotypic differences. Therefore, R. argillacea is now considered a species complex comprising four distinct species: R. argillacea sensu stricto, R. piperina, R. aegroticola, and R. eburnea (8).Infections caused by species of the R. argillacea complex have been reported in various clinical contexts in human. Chronic granulomatous disease (CGD) (9, 10, 11) and cystic fibrosis (CF) (12,13,14,15,16,17) are the major underlying clinical conditions. Rasamsonia infections may also occur in bone marrow transplant recipients (18, 19) but also may occur in the absence of any predisposing factors, as evidenced by the pulmonary and aortic graft infection reported by Doyon et al. (...