Predicting prognosis in prostate carcinoma remains a challenge when using clinical and pathologic criteria only. We used an array-based DASL assay to identify molecular signatures for predicting prostate cancer relapse in formalin-fixed, paraffin-embedded (FFPE) prostate cancers, through gene expression profiling of 512 prioritized genes. Of the 71 patients that we analyzed, all but 3 had no evidence of residual tumor (defined as negative surgical margins) following radical prostatectomy and no patient received adjuvant therapy following surgery. All of the 71 patients had an undetectable serum PSA following radical prostatectomy. Follow-up period was 44+/-15 months. Highly reproducible gene expression patterns were obtained with these samples (average R(2)=0.99). We identified a panel of 11 genes that correlated positively and 5 genes that correlated negatively with Gleason grade. A gene expression score (GEX) was derived from the expression levels of the 16 genes. We assessed the prognostic value of these genes and found the GEX significantly correlated with disease relapse (p=0.007). These results suggest that the approach we used is effective for expression profiling in heterogeneous FFPE tissues for cancer diagnosis/prognosis biomarker discovery and validation.