Background: UC is characterized by extensive genomic heterogeneity. Access to genomic DNA from all metastatic lesions is infeasible. Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) may recapitulate heterogeneity and offer an opportunity for continuous tracking of tumor evolution. Methods: We analyzed a cohort of advanced UC patients with serial (2 time points) ctDNA NGS using Guardant360. Restaging scans were examined to determine the relationship between ctDNA dynamics and radiologic progression. We performed whole exome sequencing (WES) of a subset of the corresponding tumors to define patterns of genomic heterogeneity. Results: NGS was performed on 214 individual ctDNA samples from 78 advanced UC patients (61 M, 17 F). A minimum of 2 serial ctDNA tests per patient (range 2-8) were collected over an average 21.5 (2-108) weeks between samples. Molecular alterations (MAs) were identified in 188 (88%) of samples with a mean of 4.3 alterations (1-31) per sample. 184 (85%) samples harbored SNVs, 30 (14%) harbored indels and 36 CNVs (17%). Most commonly mutated genes were TP53 (18%), ARID1A, NF1 (4.5% each), EGFR (3.5%), FGFR3 (3.4%), ERBB2 and PIK3CA (3.4% each). The most frequently amplified genes were ERRB2 and CCNE1. Serial analysis of maximum variant allele frequency (mVAF) revealed a mean 7.5-fold change between 1^st and 2^nd and a 6-fold change between 2^nd and 3^rd ctDNA samples. Interestingly, the mean rate of mVAF fold change/week was stable between serial testing time points (0.35, 0.32 p = 0.7). We observed that patients with higher initial mVAF ( > 3%) experienced a significantly larger mean fold decrease compared to patients with initial mVAF below this threshold (p = 0.008). In patients with available restaging scans timed with ctDNA testing, all patients with radiologic progression exhibited increasing mVAF (mean: 8-fold). Interestingly, ctDNA identified several clinically-significant somatic MAs not present on matching tumor WES including PIK3CA (T727R, M1043I), TP53 (Q331*, P190L), RB1 (R556*, Q257*), APC (D2527H), and BRCA2 (P2804S).WES is ongoing in more patients. Conclusions: ctDNA sequencing enables dynamic monitoring of therapy-driven clonal evolution patterns of advanced UC.