Prostate cancer is histologically and molecularly heterogeneous. Clinically significant disease is often driven by dominant intra-prostatic lesions (IPLs). Prostate cancers cluster into molecular phenotypes with substantial genetic heterogeneity making pathway-based molecular analysis appealing. MRI/ultrasound fusion biopsy provides a unique opportunity to characterize tumor biology of discrete lesions at diagnosis. This study determined the feasibility of pathway-based gene expression analysis of prostate biopsies and characterized cancer pathway deregulation.Thirteen patients had prostate cancer diagnosed by MRI/ultrasound fusion biopsy and either Gleason 6 or Gleason ≥8. Gene expression profiling was performed on 14 biopsies using >700 genes representing 13 cancer pathways. Pathway-based analysis compared gene expression among samples based on clinical, pathological, and radiographic characteristics. Pathway-based gene expression analysis was successful in 12 of 14 (86%) samples. Samples clustered based upon deregulation of DNA Repair and Notch, Chromatin Modification and Cell Cycle, or all other pathways, respectively. DNA Repair demonstrated the greatest differential deregulation. Lesions with Gleason ≥8, PSA ≥10, or intense dynamic contrast enhancement (DCE) had significantly higher DNA Repair deregulation than those with Gleason 6, PSA <10, or low to moderate DCE. Alterations in DNA Repair gene expression were diverse with upregulation of markers of DNA damage and down-regulation of DNA Repair proteins. This study demonstrates the feasibility of pathway-level gene expression analysis of discrete intra-prostatic lesions sampled by MRI/ultrasound fusion biopsy. IPLs cluster into distinct molecular phenotypes, the most significantly altered being DNA Repair.