In this study we evaluated the clinical usefulness of identifying urothelial cells with increased DNA damage with the alkaline comet assay and compare it with voided urine cytology for the assessment of markers indicative of bladder cancer. The analysis was carried out on 105 subjects having clinical suspicion of bladder cancer, and who had undergone cytology for the first time. Urine cytology and alkaline comet assay were performed on the same fresh urine samples obtained from each patient. The subjects were divided according to negative or positive cytology. The Mann-Whitney U-test showed that the comet parameters (tail moment, tail length, and % of DNA in the tail) and the numbers of comets (cells with an arbitrary cut-off value of head intensity <90% of DNA content) in subjects positive in both tests were significantly higher than in the negative group. Sensitivity, specificity, and positive and negative predictive value of the comet assay were compared with those of cytology, which is regarded as the gold standard. Sensitivity was 71.4%, specificity was 91.8%, positive and negative predictive values were 38.5 and 97.8, respectively. Two subjects negative in the comet assay were positive in cytology. Eight patients were positive in the comet assay and negative for cytology. Interestingly, one of these eight patients was later found positive for cytology. Logistic regression analysis indicates that the tail moment is significantly associated with an increased risk for positive cytology.