Background: HER2-low breast cancer is defined as HER2 IHC score of 1+ or 2+/ISH- tumors. Anti-Her-2 therapies such as trastuzumab deruxtecan improve progression-free survival (PFS) and overall survival (OS) in patients with HER-2 low metastatic breast cancer. However, the mutational landscape of HER2-low BC is still not well defined. The aim of this study is to characterize the biological and clinicopathological landscape of HER2-low BC by next generation sequencing. Methods: We conducted a single institution, retrospective chart review of patients with metastatic breast cancer (MBC) from 1/2020 to 4/2023. We obtained Next generation sequencing data and correlated it with clinical outcomes. Patients with HER2 IHC score of 1+ or 2+/ISH- tumors referred as HER2-low, while with no HER2 IHC staining at all were referred as HER2-zero, and pts with HER2 IHC 3+ or HER2 IHC 2+/ISH+ referred as HER2+. Results: A total of 98 pts were enrolled:45 pts with HER2-low tumors, 29 pts with HER2+ and 24 pts with HER2-zero tumors. In the HER2-low cohort, 58% (26 pts) were initially diagnosed with loco regional disease while 42% presented with stage IV at diagnosis. On average patients were on 3 lines of therapy for metastatic disease (ranging between 1 to 12). Of those 35% received T-DXd. High Ki-67 proliferation (≥20), reported in 58% (26 pts). OS was 54.4 months, 95% CI [ 42.5,100.2]. In the HER2+ cohort, 31% of pts presented with stage IV at diagnosis. On average pts received 3 lines of therapy (ranging from 1 to 14). 69% of pts received T-DXd. High Ki-67 reported in 48% of pts. OS was 59.2 months, 95% CI [39.2,96,1]. In the HER2-zero, 62.5% of pts presented with stage IV. On average pts received 1 line of therapy (ranging between 1 to 5), 62% of pts had high Ki-67. OS of 41.6 months 95% CI [18.4, 77.9]. Conclusions: Although limited by the sample size, our data suggests that HER-2 low breast cancer tends to be more locally advanced or metastatic at the time of presentation. HER-2 low BC is associated with a higher Ki-67 index and tends to harbor more mutations. HER-2 low may not just be a target, but a unique biological entity and can be distinguished from HER2-Zero BC with higher prevalence of HR+ and other biomarkers and better OS. NGS data from a larger data set is required to better characterize HER-2 low breast cancer. We suggest that clinicians obtain NGS panel in all patients with HER-2 low MBC.