We evaluated the minimum core number for better index tumor detection to determine the best core site as well as biopsy Gleason score heterogeneity in the same index lesion. The aim was to optimize the highest Gleason score detection. A total of 327 patients with negative digital rectal examination underwent magnetic resonance imaging/transrectal ultrasound fusion targeted biopsy for elevated/rising prostate specific antigen and/or 1 or more detectable lesions on multiparametric magnetic resonance imaging after a previous negative standard biopsy. Depending on the diameter of each index lesion (8 or less, or greater than 8 mm) 4 or 6 cores, respectively, were taken according to a well determined sequence. Of the patients 166 (50.7%) had prostate cancer, including 79 (47.6%) with an 8 mm or less index lesion and 87 (52.4%) with a greater than 8 mm index lesion. Of patients with an index tumor 8 mm or less 7 (8.9%) had 1, 31 (39.2%) had 2, 27 (34.2%) had 3 and 14 (17.7%) had 4 positive cores. Similarly, of patients with a lesion greater than 8 mm 8 (9.2%) had 1, 30 (34.5%) had 2, 13 (14.9%) had 3, 14 (16.1%) had 4, 12 (13.8%) had 5 and 10 (11.5%) had 6 positive cores. The major prevalence of positive cores was observed in the center of the target. Gleason score heterogeneity was found in 12.6% of those with an 8 mm or less target vs 26.4% with a target greater than 8 mm. In the center of the target there was a slight prevalence of Gleason pattern 4 or greater, or a lesser pattern. Approaching magnetic resonance imaging/transrectal ultrasound fusion targeted biopsy with a single core might be inadequate. Rather, taking 2 cores in the center of the index lesion may provide more accurate cancer detection and optimize the chances of finding the highest Gleason pattern.