309 Background: Regardless of the anatomic location (renal pelvis or ureter (upper tract), bladder or urethra) urothelial carcinoma is considered a single entity. Outcomes differ depending on site of initial diagnosis. Moreover, little is known about the comparative biology across primary sites. To define the genomic profile of urothelial carcinoma arising from the upper tract, we used a next generation sequencing technology to analyze 26 sporadic high-grade urothelial tumors of the renal pelvis. Methods: Frozen tumor samples and matched germline blood or normal kidney DNA from 26 nephroureterectomy specimens were obtained under an IRB-approved protocol. DNA was analyzed using a next generation, targeted sequencing assay. We compared these tumors to a set of 108 bladder tumors. Fisher’s exact test analyzed associations with pathological stage and Cox regression with log-rank p-values examined associations with bladder recurrence and disease-free survival. Fisher’s exact test was used to compare mutation frequencies between upper tract and bladder tumors. P-values were adjusted for multiple comparisons using the Benjamini-Hochberg method. Results: Surgical pathology was Ta, T1, T2 and T3 in 7 (26.9%), 5 (19.2%), 4 (15.4%) and 10 (38.5%) patients, respectively. High-grade disease was found in 24 (92.3%) patients with 6 (27.3%) patients having node positive disease. The most frequently altered genes included FGFR3 (42.3%), KDM6A (38.5%), MLL2 (26.9%), TSC1 (26.9%), CREBBP (23.1%), CDKN2A (19.2%) and HRAS (19.2%). No significant differences in tumor and patient characteristics were found between the bladder and upper tract cohorts. A significant difference in the frequency of alterations between the upper tract and bladder cohorts was found in HRAS (19.2% vs 2.8%, p=0.032), TP53 (7.7% vs 57.4%, p<0.001) and TSC1(26.9% vs 5.6%, p=0.024). Conclusions: We identified unique patterns of genomic alterations within upper tract urothelial carcinoma compared to bladder cancers. Upper tract tumors are characterized by a higher frequency of activating FGFR3 and HRAS mutations, TSC1 nonsense mutations, and a lower frequency of TP53 alterations. These findings may have future implications on the utility of targeted therapies in this disease subtype.