To determine the square measure threshold of prostate cancer lesions in pathological specimens showing PI-RADS categories 3 to 5, and to identify the pathological characteristics of cancerous lesions over the threshold. Cancer foci detected in horizontal sections of specimens were defined as pathological cancerous lesions, in which square measure, lesion location (peripheral or transition zone), Gleason pattern (GP), GP4-5 component percentages, and GP 4 subtypes were assessed. A receiver operating characteristic curve was used to determine the threshold of the square measure of pathological specimens that distinguishes between lesions of PI-RADS categories 1 and 2 and those of 3 to 5. Univariable and multivariable analyses were performed to determine the histopathological features associated with PI-RADS categories 3 to 5. A total of 100 consecutive patients underwent multiparametric magnetic resonance imaging before robotic-assisted laparoscopic prostatectomy. A total of 1366 pathological cancerous lesions were detected, 217 of which were classified as PI-RADS categories 3 to 5. A square measure of 40 mm2 on pathological specimens was the threshold for PI-RADS categories 3 to 5. Of the 415 lesions that were over 40 mm2, 211 lesions exhibited PI-RADS categories 1, 2 and 204 lesions exhibited PI-RADS categories 3 to 5. Multiple logistic regression analysis showed that square measure, fused glands, and cribriform glands were independently associated with PI-RADS categories 3 to 5. Cancerous lesions over 40 mm2 showing PI-RADS categories 3 to 5 are associated with square measure, fused glands, and cribriform glands.