Objectives To assess the degree of upgrading and increase in clinical risk category of transperineal template biopsy (TTB) compared with transrectal ultrasonography-guided prostate biopsy (TRUSB). Upgrading of TRUSB Gleason grade and sum after radical prostatectomy (RP) is well recognised. TTB may offer a more thorough mapping of the prostate than TRUSB, as well as a more accurate assessment of the tumour. In this retrospective cohort study of prospectively collected data, we compare the initial TRUSB and TTB Gleason grade and sum with the final assessment at RP. Patients and Methods Following Ethics Committee approval, 431 laparoscopic and robotic RP specimens of two urologists, fellowship-trained in minimally invasive RP, were examined in the private sector between April 2009 and October 2013. Final RP Gleason grade and sum were compared with the initial prostate biopsy. All pathological assessments were performed by a dedicated uropathology unit, experienced in prostate pathology. Upgrading was defined either as an increase in the primary Gleason grade, or as identification of a higher grade tertiary pattern at final RP analysis. Increase in clinical risk category was defined as an increase from low- (Gleason ≤6), to either intermediate- (Gleason 7) or high-risk disease (Gleason 8–10); or as an increase from intermediate- to high-risk disease. The chi-squared test was used to compare categorical variables, while the Wilcoxon rank sum was used for continuous quantitative variables. Results The 431 RP specimens comprised 283 in which the prostate cancer was diagnosed at TRUSB and 148 diagnosed at TTB. There was no difference between TRUSB and TTB in mean prostate weight (46.4 vs 44.2 g), final RP pathological stage (pT2: 187 vs 102; pT3 97 vs 48; P = 0.65) or mean tumour volume (2.15 vs 2.14 mL). Overall, 33.22% of TRUSB and 30.41% of TTB were upgraded, which was not significantly different (P = 0.55). Similarly there was no difference in whether there was an increase to a higher Gleason sum (TRUSB 23.3% vs TTB 20.9%; P = 0.57). TTB was more reflective of the actual clinical risk category, with TRUSB more likely to show an increase in clinical risk (TRUSB 22.3% vs TTB 14.2%; P = 0.04). Conclusions In this series, TTB more accurately predicted clinical risk category than TRUSB. TTB should be considered before active surveillance, to ensure that occult higher risk disease has not been under diagnosed. Upgrading and increase in clinical risk category was relatively common in each group highlighting the need for improved pretreatment staging accuracy.