The purpose of this study is to compare diagnostic performance of Prostate Imaging Reporting and Data System (PI-RADS) version (v) 2.1 and 2.0 for detection of Gleason Score (GS) ≥ 7 prostate cancer on MRI. Three experienced radiologists provided PI-RADS v2.0 scores and at least 12 months later v2.1 scores on lesions in 333 prostate MRI examinations acquired between 2012 and 2015. Diagnostic performance was assessed retrospectively by using MRI/transrectal ultrasound fusion biopsy and 10-core systematic biopsy as the reference. From a total of 359 lesions, GS ≥ 7 tumor was present in 135 lesions (37.60%). Area under the ROC curve (AUC) revealed slightly lower values for peripheral zone (PZ) and transition zone (TZ) scoring in v2.1, but these differences did not reach statistical significance. A significant number of score 2 lesions in the TZ were downgraded to score 1 in v2.1 showing 0% GS ≥ 7 tumor (0/11). The newly introduced diffusion-weighted imaging (DWI) upgrading rule in v2.1 was applied in 6 lesions from a total of 143 TZ lesions (4.2%). In summary, PI-RADS v2.1 showed no statistically significant differences in overall diagnostic performance of TZ and PZ scoring compared to v2.0. Downgraded BPH nodules showed favorable cancer frequencies. The new DWI upgrading rule for TZ lesions was applied in only few cases.