In the United States prostate cancer (PCa) is one of the most commonly diagnosed cancers and estimated to affect 239,000 men in 2013. Although few prognostic biomarkers are currently used in the clinical setting, biomarkers that predict the risk of disease aggressiveness leading to prostate specific antigen (PSA) failure after prostatectomy may assist clinicians in selecting adjuvant therapies following prostatectomy. In studies utilizing co-registered pre-operative imaging data with post-operative pathology data, validated prognostic biomarkers may also be highly useful as “gold standards” against which to train imaging methods to assess disease aggressiveness pre-operatively. We previously identified 33 potentially prognostic biomarkers from the analysis of multiple gene expression datasets and current literature. Using tissue microarrays containing PCa tissue from 170 prostatectomy specimens from men with detailed PSA and follow up data, we quantified immunohistochemical (IHC) staining for these markers within selective tissue sub-regions (malignant epithelium) and identified 16 of the 33 biomarkers which were associated with shorter time to PSA failure. While our aim is to use validated biomarkers to pre-operatively train in vivo imaging methods, current imaging capabilities have lower resolution than digital histology and it is unclear whether biomarker expression in total tumor areas (encompassing malignant epithelium, benign epithelium, stroma, and other tissue structures) which are resolvable by imaging methods will also be prognostic. In this study, we have re-evaluated our list of 16 strong candidate biomarkers to determine if IHC expression within total tumor areas are associated with PCa aggressiveness (PSA failure following prostatectomy) using our TMA cohort.