Background <p>Immunohistochemistry (IHC) is widely used to determine estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status, which are crucial for predicting breast cancer recurrence and guiding adjuvant therapy decisions. While RNA expression levels, assessed by microarray, can also determine ER and HER2 status, they do not match IHC assessment results perfectly. This study investigated the discordance rates between IHC and RNA expression-based assessment of ER and HER2 status and evaluated the prognostic impact of such discrepancies.</p> Methods <p>IHC/FISH results for determining ER/HER2 status were compared with RNA expression-based assessment. Recurrence-free survival (RFS) analysis was performed to evaluate the association between discordant results and clinical outcomes. Inverse Probability of Treatment Weighting (IPTW) was applied to adjust for potential confounding by differences in patient characteristics. Multivariate analysis was conducted to identify factors most strongly associated with ER/HER2 discordance.</p> Results <p>The discordance rate was 11.8% for ER and 19.4% for HER2 when comparing IHC/FISH with RNA expression-based assessment. Analysis of RFS suggested a possible association between discordant ER results and shorter RFS, although this did not reach statistical significance in unadjusted analysis. Using IPTW, discordance in both ER and HER2 status was found to be significantly related to worse RFS (<i>p</i> = 0.001 and <i>p</i> = 0.04, respectively). In multivariate analysis, PR-negative/HER2-positive cases were most strongly associated with ER/HER2 discordance.</p> Conclusions <p>ER and HER2 RNA expression levels should be considered for confirmation in patients with PR-negative/HER2-positive status, as discordance between IHC and RNA-based assessment is significantly associated with worse recurrence-free survival outcomes.</p>