Abstract Urothelial carcinoma (UC) of the bladder is a heterogeneous disease and presents a significant challenge in disease management. The divergence in clinical behaviour of tumors of similar stage and grade indicates the presence of molecular subgroups. In this study we aimed to identify novel genomic subgroups of UC to provide improved subclassification at diagnosis. We assessed 160 bladder tumors comprising all stages and grades and including 49 high-stage T1 (T1G3) tumors for genome-wide copy number alterations and mutations in 8 key genes (AKT1, FGFR3, HRAS, KRAS, NRAS, PIK3CA, TP53, TSC1) implicated in UC. This revealed the existence of potential genomic subclasses of the “gold-standard” stage/grade groups of UC. The clinical management of patients with T1G3 tumors is particularly challenging as this subset of tumors exhibit highly divergent behaviour. Three major subgroups of T1G3 tumors were identified that differed with respect to copy number alterations and to FGFR3 and TP53 mutation status. One of these, which showed a prevalence of copy number losses, had the highest rate of stage progression/metastasis. We also identified novel regions of copy number alteration, concomitant molecular events, and relationships between molecular events and clinico-pathological features. Tumors harbouring FGFR3 mutations were found to be more chromosomally stable than their wild-type counterparts and FGFR3 mutation was mutually exclusive with overrepresentation of 8q in non-muscle-invasive tumors. In muscle-invasive (MI) tumors, losses of regions on 10q (including PTEN), 16q and 22q and gains on 10q, 11p, 12p, 19p and 19q were positively associated with metastasis. TP53 mutation in MI tumors was positively associated with losses on 16p, 2q, 4q, 11p, 10q, 13q, 14q, 16q and 19p and gains on 1p, 8q, 10q and 12q. Molecular alterations involving chromosome 9 were highly complex and copy number loss in the CDKN2A region was positively associated with deletions on 6p, 6q, 9p, 9q, 11p, 13q and 17p, and gains on 1q, 4q, 7p, 7q, 8p, 17q and 20q in MI tumors. Our findings add information to conventional histopathological classification and may lead to improved biological understanding and the development of robust prognostic biomarkers. Studies of larger numbers of tumors are underway to confirm the subgroups and relate findings to clinico-pathological data. Citation Format: Carolyn D. Hurst, Fiona M. Platt, Claire F. Taylor, Margaret A. Knowles. Subgroups of urothelial carcinoma of the bladder defined by genomic analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 821. doi:10.1158/1538-7445.AM2013-821