Urothelial carcinoma (UC) is the most common type of cancer of the bladder and upper urinary tract, and is characterized by a high risk of recurrence and progression. Urine fluorescence in situ hybridization (FISH) is a technique that detects genetic aberrations in exfoliated cells in the urine, with specific probes for chromosomes 3, 7 and 17 and the p16 gene. To evaluate the diagnostic value of FISH in UC, 119 patients from November 2010 to June 2012 with suspected UC were recruited into a prospective, cross-sectional study and were followed up for 12-30 months. These patients received voided urine cytology and FISH tests, and underwent cystoscopy and/or ureteroscopy as a reference standard. The final diagnoses confirmed 73 patients with UC, located in the bladder, upper urinary tracts or the two. The sensitivity of FISH for detecting UC was superior to cytology, irrespective of tumor grade and stage: Overall, 80.8 vs. 32.9% (P<0.001); low grade, 75.8 vs. 12.1% (P<0.001); high grade, 85 vs. 50% (P<0.005); non-muscle-invasive, 81.1 vs. 28.3% (P<0.001) and muscle-invasive, 80 vs. 45% (P<0.05), respectively. The specificities of the two tests were similar; overall, the specificity was 89.1% for cytology vs. 100% for FISH, and no significant difference was observed between the methods. Notably, FISH exhibited 100% sensitivity for cytologically non-diagnostic UC, but 33.3% specificity. In conclusion, FISH is a reliable and non-invasive diagnostic tool for bladder and upper urinary tract UC, particularly in patients with low-grade or early stage tumors.