Background: Urothelial carcinoma of the bladder (UCB) exhibits one of the highest somatic mutation burdens. Circulating cell-free DNA (cfDNA) is obtained non-invasively from peripheral blood and appears detectable in most patients (pts) with advanced UCB. We report cfDNA profiling of patients with advanced UCB using biopsy-free cfDNA sequencing. Methods: Pts with advanced UCB that underwent cfDNA analysis using Guardant360 were identified. A 70-gene cfDNA next generation sequencing (NGS) panel from a CLIA-licensed, CAP-accredited laboratory (Guardant Health, Inc.) offers complete exon sequencing for 29 cancer genes, critical exons in 39 genes and amplifications (16 genes), fusions (6 genes) and indels (3 genes) harvested from 10 mL of peripheral blood. Results: Of 71 patients with advanced UCB, cfDNA was detectable in 61 patients (85.9%). The median age was 70 years (range 45-89). The most common recurrent somatic mutations were in TP53 (n = 35), BRCA1/2 (n = 20), ARID1A (n = 17), FGFR2/3 (n = 17), KRAS/RAF1/BRAF/MAPK (n = 15), and NF1 (n = 12), MET (n = 10) and ERBB2 (n = 10). The most common genes with increased copy numbers were kinase genes (ERBB2, PIK3CA, EGFR, RAF1, BRAF, MET and FGFR1) and cell-cycle controlling genes (MYC, CCNE1, CDK4/6). The cfDNA somatic alteration burden appeared similar to those of other major malignancies. Serial cfDNA profiling of pts receiving chemotherapy revealed the clonal evolution of mutations in genes associated with resistance including BRCA2, NF1 and GATA3. Analysis of additional patients is ongoing in this expanding dataset. Conclusions: cfDNA was frequently detected in patients with advanced UCB, and alterations were most frequently seen in TP53, kinase genes, epigenetic modifiers and cell-cycle controlling genes, which appears similar to those previously reported from muscle-invasive UCB tumor tissue NGS. Given that cfDNA offers a non-invasive means of profiling tumor DNA, identification of the mechanisms of resistance targeted with this test should be evaluated for impact on response.