Background: ERBB2 encodes human epidermal growth factor receptor 2 (HER2), a member of the EGFR family of receptor tyrosine kinases that signal through the pro-oncogenic MAP- and PI3K-kinase pathways. ERBB2 is altered by amplification and/or overexpression in various cancers, including urothelial carcinoma (UC). These alterations could confer sensitivity to ERBB2 kinase inhibitors in selected patients with UC. Methods: Patients diagnosed with UC were enrolled onto an institutional review board approved prospective sequencing protocol. Tumor and matched germline DNA were analyzed using the MSK-IMPACT assay that detects alterations in 410 oncogenes and tumor suppressor genes, including ERBB2. Results: Overall, 449 samples from 429 patients were sequenced from 2013 to August 2016. Genetic alterations in ERBB2 were found in 78 samples (17%). At the time of sample collection, 24 patients (31%) had non-muscle invasive bladder cancer (NMIBC), 30 patients (38%) had muscle-invasive bladder cancer (MIBC), 18 (23%) had metastatic disease and 7 (8%) had upper tract urothelial carcinoma (UTUC). Sixteen samples (21%) came from metastatic specimens. Of the observed 78 ERBB2 alterations, 20 samples had amplifications (26%) and 58 samples had mutations (74%). Seven samples (9%) had both. We identified 71 missense, 2 inframe and 1 fusion alteration, corresponding to a somatic mutation rate of 13.1%. Thirty-seven mutations (50%) were functionally characterized hot spots that are potentially actionable. Of note, the hot spot mutation S310F/Y was found in 29 samples (37%). Its mutation allele frequency varied significantly. There was a trend towards a higher mutant allele frequency of S310F/Y in higher stage disease without reaching statistical significance (Table). In our cohort, 3 patients were enrolled in clinical trials with ERBB2 kinase inhibitors based upon the presence of an ERBB2alteration. Conclusions:ERBB2is a frequent mutation at different stages of UC. In higher stage disease, clonal selection of the S310F/Y hot-spot mutation may occur and requires further study.