Objective: To assess PI-RADSv2.1 and PI-RADSv2 descriptors across readers with varying experience. Methods Twenty-one radiologists (7 experienced (≥ 5 years) seniors, 7 less experienced seniors and 7 juniors) assessed 240 ‘predefined’ lesions from 159 pre-biopsy multiparametric prostate MRIs. They specified their location (peripheral, transition or central zone) and size, and scored them using PI-RADSv2.1 and PI-RADSv2 descriptors. They also described and scored ‘additional’ lesions if needed. Per-lesion analysis assessed the ‘predefined’ lesions, using targeted biopsy as reference; per-lobe analysis included ‘predefined’ and ‘additional’ lesions, using combined systematic and targeted biopsy as reference. Areas under the curve (AUCs) quantified the performance in diagnosing clinically significant cancer (csPCa; ISUP ≥ 2 cancer). Kappa coefficients ( κ ) or concordance correlation coefficients (CCC) assessed inter-reader agreement. Results At per-lesion analysis, inter-reader agreement on location and size was moderate-to-good ( κ = 0.60–0.73) and excellent (CCC ≥ 0.80), respectively. Agreement on PI-RADSv2.1 scoring was moderate ( κ = 0.43–0.47) for seniors and fair ( κ = 0.39) for juniors. Using PI-RADSv2.1, juniors obtained a significantly lower AUC (0.74; 95% confidence interval [95%CI]: 0.70–0.79) than experienced seniors (0.80; 95%CI 0.76–0.84; p = 0.008) but not than less experienced seniors (0.74; 95%CI 0.70–0.78; p = 0.75). As compared to PI-RADSv2, PI-RADSv2.1 downgraded 17 lesions/reader (interquartile range [IQR]: 6–29), of which 2 (IQR: 1–3) were csPCa; it upgraded 4 lesions/reader (IQR: 2–7), of which 1 (IQR: 0–2) was csPCa. Per-lobe analysis, which included 60 (IQR: 25–73) ‘additional’ lesions/reader, yielded similar results. Conclusions Experience significantly impacted lesion characterization using PI-RADSv2.1 descriptors. As compared to PI-RADSv2, PI-RADSv2.1 tended to downgrade non-csPCa lesions, but this effect was small and variable across readers.