BACKGROUND AND AIM: The aim of this study was to assess the relative sensitivities and specificities of fluorescence in situ hybridization (FISH) and routine cytology for the detection of malignancy in biliary tract strictures. METHODS: Bile duct brushing and aspirate specimens were collected from 131 patients being evaluated for possible malignant bile duct strictures. Both specimen types were assessed by FISH but only brushing specimens were assessed by cytology. The FISH assay used a mixture of fluorescently-labeled probes to the centromeres of chromosomes 3, 7, and 17 and chromosomal band 9p21 (Vysis® UroVysion) to identify cells having chromosomal abnormalities. A case was considered positive for malignancy if five or more cells exhibited polysomy. RESULTS: Sixty-six of the 131 patients had surgical pathologic and/or clinical evidence of malignancy. Thirty-nine patients had cholangiocarcinoma, 19 had pancreatic carcinoma, and 8 had other types of malignancy. The sensitivity of cytology and FISH for the detection of malignancy in bile duct brushing specimens in these patients was 15% and 34% (p < 0.01), respectively. The sensitivity of FISH for the bile aspirate specimens was 23%, and the combined sensitivity of FISH for aspirate and brushing specimens was 35%. The specificity of FISH and cytology brushings were 91% and 98% (p = 0.06), respectively. CONCLUSIONS: FISH is significantly more sensitive than and nearly as specific as conventional cytology for the detection of malignant biliary strictures in biliary brushing specimens. FISH may improve the clinical management of patients who are being evaluated for malignancy in bile duct strictures.