Background: Limited studies exist comparing methods for HER2 status determination using real-world data (RWD). Results of metastatic breast cancer (MBC) patients with Next Generation Sequencing (NGS) within the ConcertAI Genome360 dataset with DNA sequencing were compared to the standard immunohistochemistry (IHC) or in-situ hybridization (ISH) or both IHC and ISH for HER2 status. Methods: Patients diagnosed with MBC, with HER2 status determined with NGS DNA sequencing, IHC, or any of the ISH tests, by various laboratories within 2 months of each other, from August 19, 2011 to March 9, 2021, were analyzed. Patients were classified as positive, negative, or equivocal based on each of the testing methods. Comparisons were made between DNA sequencing and IHC results, DNA and ISH results, and DNA and IHC and subsequent ISH results. Results: The study sample included 252 patients (99% female, mean age [SD] of 62 [11.93]) with 382 test combinations as follows: 220 (58%) with DNA sequencing and IHC results, 127 (33%) with DNA sequencing and ISH results, and 35 (9%) with DNA sequencing and IHC and ISH results. Of the 220 combinations with DNA sequencing and IHC results, 15 were uniformly considered HER2+, 160 HER2-, and 1 equivocal, while 44 differed in the results, resulting in 80% match rate. Of the 127 combinations with DNA sequencing and ISH results, 14 were HER2+ and 96 HER2-, while 17 differed, resulting in 87% match rate. Of the 35 combinations with DNA sequencing and IHC and ISH, 3 were HER2+ and 26 were HER2-, while 6 differed, resulting in 83% match rate. Match rate was significantly higher for patients with negative results, than positive results. Specimen type, site, and type of expression had no impact on match rates. Conclusions: RWD indicate relatively high level of match rate among DNA sequencing, IHC, and ISH results in MBC patients. However, 13-20% of the combinations did not match. For these patients, mechanisms to reach consensus need to be explored to determine optimal treatments.