This study was to compare the concordance of transcription-quantitative polymerase chain reaction (RT-qPCR) with immunohistochemistry (IHC) in determining estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and tumor proliferation index (Ki67) status in breast cancer, and to assess the prognosis based on different subtypes. Totally 323 breast cancer patients were selected, including 216 in the training set and 107 in the validation set. Logistic regression models were constructed using 5-fold cross-validation with the mRNA expression of each biomarker as the predictor and the corresponding IHC expression level as the binary response variable. Receiver operating characteristic curve was used to determine the cutoff value. When the thresholds of ER, PR, HER2, and Ki67 were 0.764, 0.709, 0.161, and 0.554, there existed high concordance rates between IHC and RT-qPCR in ER (94.4%), PR (88.0%) and HER2 (89.4%) and a medium concordance rate in Ki67 (67.8%), which were further confirmed in the validation set (ER: 81.3%, PR: 78.3%, HER2: 80.4%, and Ki67: 69.1%). Based on the subtyping stratified by RT-qPCR, the 5-year recurrence-free interval rates of patients with luminal, HER2-enriched, and triple-negative subtypes were 88% (95% CI: 0.84-0.93), 82% (95% CI: 0.73-0.92) and 58% (95% CI: 0.42-0.80), respectively, which were similar to those assessed by IHC (88%, 78% and 47%). RT-qPCR may be a complementary method to IHC, which can not only provide additional useful information in clinic, but also show more advantages over IHC in determining certain subtypes of breast cancer.Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.