© 2018, Macmillan Publishers Limited, part of Springer Nature. Background: Magnetic resonance imaging (MRI)/ultrasound (US) fusion-guided biopsy has improved the ability to localize and detect prostate cancer (PCa) with efficiency surpassing systematic biopsy. Nevertheless, some patients have PCa missed using the MRI-targeted biopsy sampling alone. We aim to identify clinical and imaging parameters associated with cases where targeted biopsy did not detect PCa compared to systematic biopsy. Methods: We conducted a retrospective review of patients who underwent MRI/US fusion-guided biopsy in addition to concurrent systematic, extended-sextant biopsy between 2014 and 2017. For patients with PCa detected on systematic biopsy not properly localized by MRI/US fusion-guided biopsy, the sextant distance from MRI-targeted lesion to the cancer-positive sextant was calculated and parameters potentially predicting this targeting miss were evaluated. Results: In all, 35/127 (27.6%) patients with single-session MRI/US fusion-guided biopsy plus standard biopsy finding PCa had lesions incorrectly localized. Of these, 15/35 (42.9%) were identified as possible fusion-software misregistrations. The remainder, 12/35 (34.3%), represented targeted biopsies one sextant away from the cancer focus and 8/35 (22.9%) targeted biopsies two sextants away from the cancer focus. Only 7/35 (20.0%) patients were determined to have clinically significant PCa, which represents 7/127 (5.5%) of the overall population. Lower MRI lesion volumes (p = 0.022), lesion density (p < 0.001), and PI-RADS scores (p < 0.001) were significantly associated with targeted biopsy missing PCa detected on systematic biopsy. Conclusion: Clinically significant PCa is rarely missed utilizing MRI/US fusion-guided biopsy. With the majority of missed tumors representing targeting misregistrations or cases of low-grade cancer in sextants immediately adjacent to MRI suspicious lesions. Lower MRI lesion volumes, lesion density, and PI-RADS are predictors of cases with targeted biopsies missing cancer, for which systematic sampling of the sextants containing MRI targets and adjacent sextants would most optimize PCa detection.