Objective: To compare detection rates of clinically significant prostate cancer (Gleason 7 or above) between standard transrectal ultrasound guided biopsy (TRUSGB) and software-assisted systematic biopsy (SASB) using magnetic resonance/ultrasound fusion in the setting of negative multiparametric magnetic resonance imaging (mpMRI) in biopsy-naïve patients. Methods: We reviewed our prospectively maintained database of consecutive men that underwent prostate biopsy following mpMRI from September 2015 to December 2016. Patients with no prior biopsy (ie, biopsy naïve) whose mpMRI demonstrated no targetable lesions were included. Patients underwent either TRUSGB (n = 23) or SASB (n = 29). SASB was performing using the MRI/US fusion systematic template. Prebiopsy data were collected, and the detection rate of clinically significant prostate cancer was compared between biopsy approaches. Results: Baseline patient characteristics were similar between the TRUSGB and SASB groups. Clinically significant prostate cancer was found in no patients undergoing TRUSGB and in 6 patients undergoing SASB (0 vs 21%, P =.028). Conclusion: Based on our institutional experience, SASB is associated with a higher detection rate of clinically significant prostate cancer when compared to TRUSGB for biopsy-naïve patients with negative prostate mpMRI. Software co-registration of the MRI and ultrasound image may optimize the distribution of biopsy cores allowing for improved prostate cancer detection compared to conventional TRUSGB.