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. Blood was collected preoperatively on the day of surgery, and plasma and buffy coat were processed for extraction of ctDNA. FFPE samples from RNU were used for tissue genomic DNA extraction. Next-generation sequencing (NGS) was used for variant profiling. Plasma and somatic tissue mutations were called by comparing with matched buffy coat samples. Detection of cancer variants with a mutation allele frequency (MAF) ≥ 0.25% and hotspot variants with a MAF down to 0.1% were reported for plasma samples targeted by a NGS panel (PredicineCARE). Variants with a MAF ≥ 5% and hotspot variants with a MAF down to 2% were reported for FFPE samples. Results: NGS analyses of matched FFPE and plasma samples were successfully performed for all 15 accrued UTUC patients. Alterations in MYC amplification (62%), TERT promoter (62%), TP53 (38%), FGFR3 (31%), ERBB2 (25%), ARID1A (19%), and PIK3CA (19%) were demonstrated in urothelial tumor tissue. Matched plasma ctDNA showed prevalent alterations in the TERT promoter (47%), TP53 (30%), ARID1A (20%), ERBB2 (20%), FGFR3 (20%), and PIK3CA (17%). Five patients (33%) had detectable plasma ctDNA mutations concordant with tumor-based genotypes 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 71.4% sensitivity. The panel was 100% specific with no patients with pTis, pTa, or pT1 and pN0 having detectable concordant ctDNA mutations. Conclusions: Prospective ctDNA analysis using a targeted NGS panel is a feasible nonsurgical approach to prediction of high-risk UTUC and has the potential for identification of upper tract urothelial cancer patients that may benefit from NAC.