The purpose of this study was to develop a multitarget, multicolor fluorescence in situ hybridization (FISH) assay for the detection of urothelial carcinoma (UC) in urine specimens. Urinary cells obtained from voided urine specimens of 21 patients with UC and 9 normal donors were analyzed with nine different centromere enumeration probes and a single locus-specific indicator probe to determine an optimal set of FISH probes for UC detection. The four probes with the greatest sensitivity for UC detection were then labeled with a unique fluorophore and combined into a single probe set. The probes with the greatest combined sensitivity for UC detection were CEP3, CEP7, CEP17, and the 9p21 (P16) LSI. This probe set was used to evaluate urine specimens acquired from 179 patients for prospective testing (46 with biopsy-proven UC). FISH slides were evaluated by scanning the slide for cells with nuclear features suggestive of malignancy and assessing the FISH signal pattern of these cells for polysomy (ie, gains of two or more different chromosomes). A receiver operator characteristic curve revealed that a cutoff of 5 cells with polysomy as the positive criterion for cancer resulted in an overall sensitivity of 84.2% for patients with biopsy-proven UC and a specificity of 91.8% among patients with genitourinary disorders but no evidence of UC. This study demonstrates that a multitarget, multicolor FISH assay containing centromeric probes to chromosomes 3, 7, and 17 and a locus-specific probe to band 9p21 has high sensitivity and specificity for the detection of UC in voided urine specimens.