Background: Urothelial carcinoma (UC) is highly lethal malignancy with limited therapeutic options in the advanced setting. Using a targeted, deep sequencing approach, we analyzed high grade bladder tumors to determine the prevalence of known cancer genes and to identify potential targets for therapy. Methods: High-grade bladder UC frozen tumor and matched germline blood was obtained from 109 patients (pts) undergoing transurethral resection or cystectomy. Tumor and normal DNA were analyzed using IMPACT (Integrated Molecular Profiling of Actionable Cancer Targets), a next generation, exon-capture targeted deep sequencing assay designed to identify point mutations, indels, and copy number alterations in the coding regions of 300 cancer-associated genes. Results: Median age was 68 and 82 pts (75%) were male. Mean target coverage for all sequenced exons was 579X. The most commonly mutated gene was TP53 (63 pts, 58%). Alterations in genes regulating cell cycle were also prevalent, including mutations in Rb (25 pts, 23%), and copy number gains of CCND1 (19 pts, 17%) and CCNE1 (8 patients, 7%). Mutations in several chromatin remodeling genes were also highly prevalent, including KDM6A (46 pts, 42%), MLL2 (34 pts, 31%), and ARID1A (31 pts, 28%). Alterations within recognized cancer-related signaling pathways were common. PI3K pathway alterations were prevalent, with PIK3CA mutations found in 25 pts (23%), 23 of which were recurrent missense mutations. Six pts (6%) had mutations in PTEN. Mutations in TSC1 were also found in six pts (6%). Alterations in select MAPK pathway genes were common, including FGFR3 (21 pts, 19%) and ERBB2(15 pts, 14%). Conclusions: High-grade bladder UC is a genetically heterogenous disease. However, over half of the tumors analyzed in this study harbor alterations in genes that have been targeted for therapeutic benefit in other cancers. Results from our targeted exon-sequencing analysis therefore provide a compelling case for clinical trials using novel agents in UC. Moreover, this deep sequencing assay may be used clinically to prospectively identify pts for trials enriched for patients with specific alterations.