The most commonly applied techniques to assess human epidermal growth factor receptor 2 (HER2) expression in breast cancer are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). HER2 detection by reverse transcription quantitative polymerase chain reaction (RT-qPCR) can provide standardized, objective and automated assessment and reflect the HER2 expression continuity. Currently, there is lack of sufficient evidence to validate whether RT‑qPCR technique is more appropriate for the detection of HER2 low expression, especially ultra-low expression. Here, we primarily utilized RT-qPCR to differentiate HER2 true negative, ultra-low and 1 +, and compare the clinicopathological features and prognosis between RT-qPCR and IHC. 136 breast cancer cases with HER2 0 or 1 + were collected, also included 21 cases with HER2 2 + FISH negative as well as 25 cases with HER2 positive during the same period for comparative analysis. Compared the mRNA levels based on IHC/FISH scores. The receiver operating characteristic (ROC) curve was utilized to determine the threshold for reclassification, and the clinicopathological characteristics and prognosis differences among IHC true negative, ultra-low and 1 + after re-classification by RT-qPCR were analyzed. The mRNA level significantly differed between the IHC 0 and 1 + groups (p < 0.001). The IHC 0 group was further divided into true negative and ultra-low, there was no statistically significant difference in mRNA levels between true negative and ultra-low groups, while the difference between ultra-low and 1 + mRNA levels was statistically significant (p < 0.001). After reclassification of IHC true negative, ultra-low and 1 + by RT-qPCR, there were statistically significant differences in histological grade, ER, PR and TILs expression. There was no significant difference between DFS and OS in the two classification methods. RT-qPCR classification aids in distinguishing clinicopathological characteristics and can serve as a supplementary technique for detecting HER2-low by IHC.Copyright © 2023 Elsevier GmbH. All rights reserved.