The purpose of this article is to compare transrectal ultrasound (TRUS) biopsy accuracies of operators with different levels of prostate MRI experience using cognitive registration versus MRI-TRUS fusion to assess the preferred method of TRUS prostate biopsy for MRI-identified lesions. SUBJECTS AND METHODS; One hundred patients from a prospective prostate MRI-TRUS fusion biopsy study were reviewed to identify all patients with clinically significant prostate adenocarcinoma (PCA) detected on MRI-targeted biopsy. Twenty-five PCA tumors were incorporated into a validated TRUS prostate biopsy simulator. Three prostate biopsy experts, each with different levels of experience in prostate MRI and MRI-TRUS fusion biopsy, performed a total of 225 simulated targeted biopsies on the MRI lesions as well as regional biopsy targets. Simulated biopsies performed using cognitive registration with 2D TRUS and 3D TRUS were compared with biopsies performed under MRI-TRUS fusion. Two-dimensional and 3D TRUS sampled only 48% and 45% of clinically significant PCA MRI lesions, respectively, compared with 100% with MRI-TRUS fusion. Lesion sampling accuracy did not statistically significantly vary according to operator experience or tumor volume. MRI-TRUS fusion-naïve operators showed consistent errors in targeting of the apex, midgland, and anterior targets, suggesting that there is biased error in cognitive registration. The MRI-TRUS fusion expert correctly targeted the prostate apex; however, his midgland and anterior mistargeting was similar to that of the less-experienced operators. MRI-targeted TRUS-guided prostate biopsy using cognitive registration appears to be inferior to MRI-TRUS fusion, with fewer than 50% of clinically significant PCA lesions successfully sampled. No statistically significant difference in biopsy accuracy was seen according to operator experience with prostate MRI or MRI-TRUS fusion.