The prevalence of fungi in the respiratory tracts of cystic fibrosis (CF) patients has risen. However, fungal surveillance is not routinely performed in most clinical centers in the United States, which may lead to an underestimation of the true prevalence of the problem. We conducted a prospective study comparing the rates of detection for clinically important fungi (CIF), defined as ,, and species and, in CF sputa using standard bacterial and selective fungal culture media, including Sabouraud dextrose agar with gentamicin (SDA), inhibitory mold agar (IMA), and brain heart infusion (BHI) agar with chloramphenicol and gentamicin. We described the prevalence of these fungi in an adult CF population. A total of 487 CF respiratory samples were collected from 211 unique participants. CIF were detected in 184 (37.8%) samples. Only 26.1% of CIF-positive samples were detected in bacterial culture medium, whereas greater rates of detection for fungi were found in IMA (65.8%; < 0.001), in SDA (at 30°C, 64.7%; = 0.005), and in BHI agar (63.0%; = 0.001). The prevalences of and species were 40.8% and 5.2%, respectively, which are greater than the nationally reported prevalence numbers of 20.4% and 1.9%. Selective fungal culture media and longer incubation periods yielded higher rates of detection for CIF in CF sputum samples compared with that detected in bacterial culture medium, resulting in an underdetection of fungi by bacterial culture alone. The prevalence of fungi in CF may be better estimated by using selective fungal culture media, and this may translate to important clinical decisions.