301 Background: Urothelial carcinoma is characterized by multifocal and metachronous tumors. To explain this phenomenon, two hypotheses have been proposed: the ‘field defect’ hypothesis - urothelial cells are primed to undergo transformation by exposure to carcinogens, and the clonal, or ‘single progenitor cell,’ hypothesis - tumors arise from intraluminal seeding of transformed cells. Methods: To examine their clonal relationships, we compared the genomic profiles of primary upper tract urothelial carcinoma (UTUC) tumors and metachronous bladder tumors (intravesical recurrences) in patients treated with radical nephroureterectomy (RNU) and subsequent transurethral resection. Specimens were analyzed using a next-generation, targeted sequencing assay designed to identify point mutations, indels, and copy number alterations in 341 cancer-associated genes. Results: We analyzed 16 primary UTUC tumors and 41 intravesical recurrences in patients treated with RNU. The median number of intravesical recurrences per patient was 2 (range 1-7) and the interval from RNU to intravesical recurrence ranged from 3.5 months to 129 months. With an average sequencing coverage of 516x, we found strong evidence delineating the clonal relationship between primary UTUC tumors and subsequent bladder tumors. The majority of somatic mutations present in the primary UTUC tumors (median=7, range 4-39) were detected in all subsequent bladder tumors (128/146, 88%). In an illustrative case, one patient followed with periodic cystoscopy/cytology who had been NED for 5.5 years then developed 7 bladder tumors over the next 44 months, each with the identical mutation profile (8 mutations) as the primary tumor. Conclusions: We demonstrate that bladder tumors following RNU represent true intravesical recurrences, with almost all tumors sharing the same somatic mutation profile as the primary UTUC tumor. This has important implications for surgical techniques to minimize the risk of intraluminal seeding, the delivery of intravesical therapy following RNU, and the development of strategies employing systemic chemotherapy or targeted agents.