To evaluate the ability of fluorescence in situ hybridization (FISH) in detecting bladder urothelial carcinoma (BUC), FISH and cytology were compared for the evaluation of 308 consecutive urine samples from patients suspected of having BUC. All patients underwent cystoscopy for identification of bladder lesions. The FISH results were compared with the cytology assessment. In all, 122 patients had confirmed BUC. Among them, 68 (55.7%) were FISH-positive, while only 33 (27%) were positive on cytology. According to disease stage (superficial vs. invasive) and grade (low vs. high), the sensitivities of FISH were also significantly higher than those of cytology in all categories. Moreover, in 36 patients who had no visible tumor with flat, erythematous mucosa (suspicious lesion), FISH was more sensitive than cytology for the detection of BUC (83.3% vs. 33.3%, P=0.002). The FISH was negative in 168 (90.3%) of 186 patients with no histological evidence of BUC or negative cystoscopy findings. The sensitivity of FISH for detecting BUC was superior to that of cytology, regardless of tumor stage and grade. FISH is a significant additional and complementary method for detection of BUC in patients who have suspicious lesions on cystoscopy.