Introduction: There are reports that the 12-core template systematic biopsies (SBx) obtained by using software registration machines (e.g., Artemis) have higher cancer detection rates (CDRs) of prostate cancer (PCa) than the standard, freehand 12-core transrectal ultrasound (TRUS)-guided biopsies. The goal of our study is to compare the clinically significant (CS) CDRs of SBx in two independent cohorts who underwent freehand TRUS-SBx alone (Cohort A) or machine-guided SBx as part of a combined MRI-ultrasound (MRI-US) fusion biopsy (FBx) (Cohort B). Materials and Methods: A retrospective review of all patients undergoing prostate biopsies over a 4-year period at the University of Cincinnati Medical Center was performed. CS cancer was defined as having a Gleason score ‡7. MRI-US FBx were obtained by using an Artemis software registration device (ARTEMISÔ, Eigen, Inc., Grass Valley, CA). Statistical significance was considered at p < 0.05. Results: Nine hundred and thirty men underwent SBx (Cohort A: 474, Cohort B: 456). There were no statistical differences between cohort A and B in CS CDRs in the overall population (39.3% vs 33.8%; p = 0.093), biopsy naive patients (40.4% vs 39.8%; p = 0.951), or patients with a prior negative biopsy (22.7% vs 25.0%; p = 0.910). Multivariate logistic regression controlling for age, race, prostate-specific antigen level, prostate volume, abnormal digital rectal exam, and family history of PCa demonstrated comparable CS CDRs, which was maintained when further stratified by prior biopsy history (all patients: odds ratio [OR] 0.99, 95% confidence interval [CI] 0.71-1.38, p = 0.958; biopsy naive: OR 0.79, 95% CI 0.51-1.22, p = 0.291; prior negative biopsy: OR 0.64, 95% CI 0.21-1.75, p = 0.403). Conclusions: Our study did not find a significant difference in the CS CDRs of machine-guided SBx compared with the freehand TRUS-SBx. Unless the SBx is done at the time of FBx, the use of these machines for obtaining SBx only is unlikely to result in any increase of CS CDRs.