Serial evaluation of circulating tumor DNA may allow noninvasive assessment of drivers of resistance to immune checkpoint inhibitors (ICIs) in advanced urothelial cancer (aUC). We used a novel, amplicon-based next-generation sequencing assay to identify genomic alterations (GAs) pre- and post-therapy in 39 patients with aUC receiving ICI and 6 receiving platinum-based chemotherapy (PBC). One or more GA was seen in 95% and 100% of pre- and post-ICI samples, respectively, commonly in TP53 (54% and 54%), TERT (49% and 59%), and BRCA1/BRCA2 (33% and 33%). Clearance of ≥1 GA was seen in 7 of 9 patients responding to ICI, commonly in TP53 (n = 4), PIK3CA (n = 2), and BRCA1/BRCA2 (n = 2). A new GA was seen in 17 of 20 patients progressing on ICI, frequently in BRCA1/BRCA2 (n = 6), PIK3CA (n = 3), and TP53 (n = 3), which seldom emerged in patients receiving PBC. These findings highlight the potential for longitudinal circulating tumor DNA evaluation in tracking response and resistance to therapy.