Objective To compare the relative value of magnetic resonance imaging (MRI) in biopsy-naive patients to those with previous negative biopsy. Although MRI-targeted biopsy has been studied in several major prostate cancer (PCa) cohorts (biopsy naive, previous negative biopsy, and active surveillance), the relative benefit in these cohorts has not been established. Methods We retrospectively reviewed biopsy-naive (n = 45) and previous negative biopsy (n = 55) patients who underwent prostate MRI prior to biopsy at our institution. Patients with an MRI suspicious region (MSR) underwent MRI-targeted biopsy as well as a systematic template biopsy, whereas those without MSR underwent only the template biopsy. All biopsies were performed with the TargetScan (Envisioneering, Pittsburgh, PA) biopsy system. MRI targeting was performed with cognitive guidance. Results On multivariate logistic regression, the presence of an MSR was the only statistically significant and independent predictor of Gleason ≥ 7 PCa on biopsy for biopsy-naive men (odds ratio [OR] 40.2, P =.01). For men with previous negative biopsy, the presence of MSR was not a predictor of Gleason ≥ 7 PCa on biopsy (OR 4.35, P =.16), whereas PSA density > 0.15 ng/mL2 was a significant and independent predictor (OR 66.2, P <.01). Conclusion Prostate MRI should be considered prior to biopsy in all patients presenting with clinical suspicion for PCa, as presence of a MSR will help guide prebiopsy counseling and provide an opportunity for MRI targeting during biopsy.