Background: Biological heterogeneity of HER2 positive breast cancers has been suggested by a modest benefit of HER2-targeted therapies reported in the APHINITY and ExteNET trials. This highlights the need for improved biomarkers that more precisely identify patients who benefit from HER2-directed agents. The 80-gene molecular subtyping signature (80GS) classifies breast tumors into Luminal, HER2 or Basal type based on the gene expression of downstream signaling pathways. Previous work showed a substantial proportion of tumors identified as HER2 equivocal or HER2 positive by 2013 ASCO/CAP guidelines may be reclassified as non-HER2 type by 80GS. In 2018, ASCO/CAP HER2 IHC/ISH classification guidelines were revised to reduce the frequency of HER2 equivocal cases, for which treatment recommendations have been ambiguous. Here we evaluated concordance between HER2 status by 2018 ASCO/CAP guideline classification and 80GS molecular subtyping. Methods: Pathology reports are provided by physicians for samples that are tested with the 70-gene risk of distant recurrence signature (70GS) and 80GS as part of routine diagnostic care. This analysis includes data sent to Agendia (Irvine, CA) from January 2019 to January 2020. HER2 IHC/ISH results based on ASCO/CAP 2018 guidelines were available for 1453 samples. Results: Of 1453 samples, 1336 (92%) were HER2 negative, 99 (7%) were HER2 positive, and 18 (1.2%) were HER2 equivocal under 2018 guidelines. 80GS reclassified 57 of 99 (58%) HER2 positive tumors as Luminal and 11 of 99 (11%) as Basal; the remaining 31% were confirmed HER2. Furthermore, 55 of 99 (55%) HER2 positive tumors were also ER and PR positive by IHC, with 48 (87%) of these reclassified as Luminal type. Of HER2 negative tumors, 80GS classified 94 of 1336 (7%) as Basal and 2 of 1336 (0.15%) as HER2. Of HER2 equivocal tumors, 16 of 18 (89%) reclassified as Luminal and 2 of 18 (11%) as Basal. Conclusions: In this real-world diagnostic dataset, 2018 ASCO/CAP guidelines resulted in few HER2 equivocal tumors overall, confirming the positive impact of the revised guidelines. However, 80GS reclassified 69% of HER2 positive tumors to non-HER2 molecular subtypes, suggesting these tumors may have suboptimal responses to HER2-directed therapy compared to HER2 enriched. All HER2 equivocal tumors reclassified to non-HER2 subtypes. Molecular classification by 80GS adds further precision in classifying HER2 positive patients and potential to predict responsiveness to HER2-targeted therapies. Further studies are warranted to validate the utility of HER2 status based on 80GS.