Abstract Background/Objectives: Determining long-term trends in tumor biomarker expression is essential for understanding aspects of tumor biology amenable to change. Limiting the availability of such data, currently used assays for biomarkers are relatively new. For example, assays for the estrogen receptor (ER), which are the oldest, extend back only to the 1970s. Methods: To extend scant knowledge about the feasibility of obtaining long-term data on tumor biomarkers, we randomly selected 60 breast cancer cases (10 per decade) diagnosed between 1947–2009 among women members of the Kaiser Permanente Northern California health plan to obtain and analyze their formalin-fixed paraffin-embedded (FFPE) tumor specimens. For each tumor specimen, we created duplicate tissue microarrays for analysis. Results: We located tumor blocks and pathology reports for 50 of the 60 cases (83%), from which we randomly sampled 5 cases per decade for biomarker analysis (n=30). All 30 cases displayed excellent morphology and exhibited biomarkers compatible with histologic type and grade. Test–retest reliability was also excellent: 100% for ER; 97% for human epidermal growth factor receptor 2 and epidermal growth factor receptor; 93% for progesterone receptor and cytokeratin 5/6; and 90% for Ki67 and molecular phenotype; the kappa statistic was excellent (>0.9) for 4 of the 7 biomarkers, strong (0.6–0.8) for 2, and fair for only 1 (owing to low prevalence). Conclusions: These results indicate immunostaining for biomarkers commonly used to evaluate breast cancer biology and assign surrogate molecular phenotypes can reliably be employed on archival FFPE specimens up to 60 years old.