Background: HER2 is an emerging prognostic and therapeutic target in uterine serous carcinoma (USC). Testing algorithms and platforms in breast and gastric cancers are well studied and validated, but optimal HER2 testing in uterine cancer is not yet established. We aimed to assess the concordance of chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), and next generation sequencing (NGS) platforms to aid in the development of USC specific testing guidelines. We also evaluated the rate of downstream mutations that may affect response to HER2 directed therapy. Methods: A total of 2,192 USC tumors were analyzed using NGS (NextSeq, 592 Genes and WES, NovaSEQ), a subset of 1,423 tumors were also tested by IHC and CISH (Caris Life Sciences, Phoenix, AZ). HER2 positivity through IHC (4B5, Ventana) and CISH (INFORM DUAL HER2 ISH Assay, Ventana) was determined based on 2007 and 2018 ASCO/CAP HER2 breast cancer guidelines. PD-L1 expression was tested by IHC using SP142 (Spring Biosciences) (positive cut-off >1%). Microsatellite instability (MSI) was tested by fragment analysis (FA), IHC and NGS. Tumor mutational burden (TMB) was measured by totaling somatic mutations per tumor (TMB-high cut-off > 10 mutations per Mb). Statistical significance was determined using chi-square. Results: Rates of HER2 positivity were comparable using the 2018 and 2007 breast cancer guidelines (19.5% vs 17.5%; p=0.25). Based on 2018 guidelines, the concordance between IHC and CISH was 98.9%. Specifically, 229/1423 patients (16%) were IHC+/CISH+, 5 patients (0.4%) were IHC+/CISH- and 11 patients (0.8%) were IHC-/CISH+ (Table). Common pathway alterations in HER2+ USC include TP53, RTK RAS, PI3K, NOTCH, chromatin remodeling and cell cycle genes. Single gene alterations in HER2+ tumors that may implicate HER2 therapy resistance (based on pathway analyses in other tumor types) included PI3K (36%), KRAS (2.6%), and PTEN (2.1%). HER2+ tumors had low immunotherapy biomarker profiles (0.3% MSI-H, 0.8% TMB, 17.1% PD-L1). Conclusions: High concordance rates were observed between CISH and IHC. Ultimately these testing platforms need to be validated by response to HER2 targeted therapies in order to develop USC specific HER2 testing guidelines.