Biliary brushing cytology (BB) to detect cholangiocarcinoma (CCA) is integral in the surveillance of patients with primary sclerosing cholangitis (PSC). Since reactive changes can mimic carcinoma, indeterminant results are frequent. Fluorescence insitu hybridization (FISH) using the UroVysion probe set has been advocated to enhance the detection of CCA. This study evaluates the performance of FISH for detecting CCA in patients with and without PSC. A query of our pathology database for atypical and suspicious BB with concurrent FISH results was performed from 2014 to 2021. FISH (using UroVysion probe set containing centromere enumeration probes to chromosomes 3, 7, and 17) was positive if at least 5 cells demonstrated polysomy. Electronic medical records were reviewed to identify patients with PSC and CCA. CCA was confirmed by pathology or clinical impression. Of the 65 patients (103 BB) in the PSC cohort, 59 patients (94 BB) without CCA and 6 patients (9 BB) with CCA were identified. 33 non-PSC patients (41 BB) with CCA were included for comparison. Positive FISH was highest in non-PSC patients with CCA (10/41 BB, 24%). Positive FISH was seen in both PSC with (1/9 BB, 11%) and without (2/94 BB, 2%) CCA. FISH positivity was lower than expected and was positive in PSC patients without CCA. These results question the clinical utility of FISH for CCA surveillance in PSC patients.