Purpose To evaluate whether core length impacts biopsy accuracy and Gleason score underestimation compared to radical prostatectomy (RP) specimens. Methods From 2010 to 2011, 8,928 cores were trans-rectal obtained from 744 consecutive patients (178 RP, 24 %), 557 by an experienced performer (>250/year) and 187 (25 %) by in-training urology residents. Prospectively analyzed variables were core length, age, prostate volume, free and total prostate-specific antigen (PSA), PSA density and free/total PSA ratio. Results Mean core length for Gleason upgrading on RP (42.7 %, n = 76) was 11.61 (±2.5, median 11.40) compared to 13.52 (±3.2, median 13.70), p < 0.001 for perfect biopsy-RP Gleason agreement (57.3 %, n = 102). In multivariate analysis, for each unit of core length increment in millimeter, the Gleason upgrading risk decreased 89.9 %, p = 0.049 [odds ratio (OR) 0.10, 95 % confidence interval (CI) 0.01-0.99]. Biopsy positivity between experienced (35.5 %) and in-training performer (30.1 %) was not significantly different (p = 0.20), with comparable mean patient age (65.1 vs. 64.1), prostate volume (52.3 vs. 50.7) and median PSA (5.2 vs. 5.1), respectively. Denoting wider variability in terms of core length, in-training performers obtained significantly larger cores for positive biopsies (11.33 ± 3.42 vs. 10.83 ± 3.68), p = 0.043, compared to experienced performer (11.39 ± 3.36 vs. 11.37 ± 3.64), p = 0.30. In multivariate analysis, PSA density (OR 1.14, 95 % CI 1.02-1.28) and age (OR 1.04, 95 % CI 1.01-1.07) were significantly associated with biopsy positivity, p = 0.021 and p = 0.011, respectively. Conclusion While core length on trans-rectal biopsy independently affects Gleason upgrading on RP specimens, performer experience has minor impact on Gleason discordance or biopsy positivity due to a sharp learning curve. © 2014 Springer-Verlag Berlin Heidelberg.