Bladder cancer patients suffer significant treatment failure, including high rates of recurrence and poor outcomes for advanced disease. If mechanisms to improve tumour cell treatment sensitivity could be identified and/or if tumour response could be predicted, it should be possible to improve local-control and survival. Previously, we have shown that radiation-induced DNA damage, measured by alkaline Comet assay (ACA), correlates bladder cancer cell radiosensitivity in vitro. In the present study we firstly show that modified-ACA measures of cisplatin and mitomycin-C-induced damage also correlate bladder cancer cell chemosensitivity in vitro, with essentially the same rank order for chemosensitivity as for radiosensitivity. Furthermore, ACA studies of radiation-induced damage in different cell-DNA substrates (nuclei, nucleoids & intact parent cells) suggest that it is a feature retained in the prepared nucleoids that is responsible for the relative damage sensitivity of bladder cancer cells, suggestive of differences in the organisation of DNA within resistant vs. sensitive cells. Secondly, we show that ACA analysis of biopsies from bladder tumours reveal that reduced DNA damage sensitivity associates with poorer treatment outcomes, notably that tumours with a reduced damage response show a significant association with local recurrence of non-invasive disease and that reduced damage response was a better predictor of recurrence than the presence of high-risk histology in this cohort. In conclusion, this study demonstrates that mechanisms governing treatment-induced DNA damage are both central to and predictive of bladder cancer cell treatment sensitivity and exemplifies a link between DNA damage resistance and both treatment response and tumour aggression. © 2013 Wiley Periodicals, Inc.