Abstract Purpose Multiparametric MRI (mpMRI) and fusion biopsy (FBx) detect more high-risk prostate cancer (PCa) and less low-risk PCa than systematic biopsy (SBx). However, there remains a small subset of patients where SBx captures higher grade disease than FBx. We aim to identify potential mechanisms for failure of FBx biopsy in detection of clinically significant (CS) PCa. Methods We reviewed a prospectively maintained database of patients undergoing mpMRI followed by FBx and SBx from 2007-2014. In patients disease upgraded to CS disease (Gleason ≥ 7) by SBx over FBx, independent re-review of MR imaging, archived biopsy imaging, and whole mount pathology, as well as needle coordinate mapping were conducted. Multivariate logistic regression analysis was performed to determine predictors for upgrading by SBx. Results Disease upgrading based on SBx over FBx occurred in 135/1003 (13.5%) patients, of which only 62 (6.2%) were to intermediate (Gleason=7) N=51, 5.1% or high risk PCa (Gleason≥8) N=11, 1.1%. On multivariate analysis, lower PSA (p <0.001), higher MRI prostate volume (p <0.001), and lower number of target cores (p=0.001) were predictors of upgrading by SBx. Main mechanisms for under-grading by FBx included mpMRI reader oversight, presence of MR invisible cancer, FBx technique error, and intra-lesion Gleason heterogeneity. Conclusions MRI and FBx rarely misses CS PCa, as only 62/1003 (6.2%) cases were upgraded to CS disease by SBx. Imaging and biopsy techniques are continually refined and further studies will help to clarify mechanisms of FBx failure and patient populations which benefit from SBx in addition to FBx.