Background: Upper tract urothelial carcinoma (UTUC) is an aggressive cancer for which use of neoadjuvant chemotherapy (NAC) is limited by suboptimal clinical staging prior to nephroureterectomy. Detection of circulating tumor DNA (ctDNA) is associated with locally advanced and metastatic urothelial carcinoma of the bladder and may help identify UTUC patients who would benefit from NAC. Here we examine the feasibility and utility of plasma ctDNA in the diagnosis of non-organ confined high-risk UTUC. Methods: Patients with high-grade cTa-T2 UTUC without radiographic evidence of metastatic disease undergoing up-front radical nephroureterectomy (RNU) were prospectively accrued for pre- and post-operative plasma collection. Blood was collected preoperatively on the day of surgery, and plasma and buffy coat were processed for extraction of cell-free DNA and genomic DNA, respectively. FFPE tumor samples from RNU were used for tissue genomic DNA extraction. Targeted next-generation sequencing (NGS) was used for variant profiling. ctDNA positivity was defined as the presence of plasma cell-free DNA variants concordant with tissue-based variants. Results: NGS analyses of matched FFPE and plasma samples were successfully performed for all 19 accrued UTUC patients. Alterations in the TERT promoter (74%), TP53 (58%), FGFR3 (53%), myc amplification (53%), and ATM (42%) were demonstrated in urothelial tumor tissue. Matched plasma ctDNA showed prevalent alterations in the TERT promoter (42%), TP53 (42%), PIK3CA (37%), ATM (32%) and CD274 (26%). Nine patients (47%) had detectable plasma ctDNA mutations concordant with tumor-specific variants using the targeted NGS panel. All patients with detectable preoperative ctDNA had advanced staging (≥pT2 or ≥pN1) and lymphovascular invasion on final pathology, resulting in a 90% sensitivity. The panel was 100% specific with no patients with pTis, pTa, pT1 and pN0 having detectable concordant ctDNA mutations. Concordant plasma ctDNA was detected in four of nine patients postoperatively. Two of three (67%) who developed metastatic disease had detectable ctDNA while neither of the two who developed non-muscle-invasive bladder recurrences did. Conclusions: Prospective ctDNA analysis using a targeted NGS panel can be used to predict muscle-invasive and non-organ-confined UTUC preoperatively. Detectable postoperative ctDNA may indicate residual disease and predate clinical recurrence.