Background: Available HER2 targeted agents have limited benefit for UC, but exceptional tumor responses are occasionally seen. HER2 expression has been tested as a biomarker of response to these drugs. Trial eligibility is often based on HER2 expression in PTs. Previous studies found moderate to high concordance of HER2 expression in PTs and synchronous regional nodes at cystectomy, but the concordance rate with distant MTs is poorly defined and further complicated by shifting definitions of HER2 positivity. Methods: Immunohistochemical (IHC) staining for HER2 (Ventana Pathway clone 4B5) was performed on PTs and matched MTs in 79 patients with UC. IHC staining was scored (0-3+) using intensity and % positive tumor cells using 2018 ASCO/CAP guidelines for breast cancer. Fisher’s Exact Test assessed association of PT and MT HER2 scores, and Cohen’s kappa statistic (κ) assessed agreement in HER2 3+ between groups. MTs were defined as synchronous if biopsied <60 days after the PT biopsy. Results: There were 67 metachronous and 12 synchronous paired biopsies; median time between metachronous biopsies was 418 days. MT biopsy sites included lymph node (30%), bone (22%), lung (18%), pelvic/peritoneal soft tissue (11%), liver (8%), brain (4%), other (6%). HER2 2+ and 3+ expression was seen in 30.4% and 12.7% of PTs and 12.7% and 7.6% of MTs, respectively. Of HER2 3+ PTs (n=10), HER2 expression in paired MTs was 0 (n=3), 1+ (n = 3), 2+ (n = 2), and 3+ (n = 2). Of HER2 3+ MTs (n=6), HER2 expression in paired PTs was 0 (n=1), 2+ (n=3), and 3+ (n=2). An association was observed between PT and MT HER2 scores (p=0.004), however there was low agreement of HER2 3+ rates between PT and MT lesions (κ = 0.17, p=0.113). Discordance was noted in both synchronous and metachronous biopsies. Conclusions: Based on the current HER2 3+ definition, HER2 overexpression is spatially and temporally discordant in UC between PTs and distant MTs. These findings suggest that both PTs and MTs should be tested for HER2 expression in future clinical trials assessing the efficacy of HER2 targeted agents in UC. The high discordance may explain previously negative therapeutic studies.