Background Magnetic resonance imaging (MRI)-ultrasound (US) fusion biopsy remains challenging and highlights the need towards standardization. Purpose To characterize the clinical and MRI features of clinically significant prostate cancer (csPCa) with discrepant Gleason score (GS) in MRI-US fusion biopsy. Material and Methods A total of 400 consecutive patients with suspected cancer lesions who underwent MRI-US fusion biopsy and subsequent prostatectomy were included. In the comparison of biopsy GS with pathology GS, matched lesions were defined as a GS, and discrepant lesions were defined as an upgrade of the GS. Descriptive statistics were used to define clinical characteristics, including age, prostate-specific antigen (PSA), PSA density, and maximal cancer core length (MCCL). Differences between lesions with matched and discrepant GS were determined considering the location and PI-RADS v2 score. A paired comparison of the volumes between the two groups was performed. Results There were 130 lesions with discrepant GS in 124 patients. There was no significant difference in the age, PSA, and PSA density between the two groups, except for the MCCL ( P = 0.028). The lesions were distributed in the peripheral (n = 88) and transition (n = 42) zones; 33, 50, and 47 lesions were at the apex, mid-gland, and base levels, respectively. PI-RADS scores were as follows: 2 (n = 5), 3 (n = 8), 4 (n = 68), and 5 (n = 39). In comparison with matched lesions, discrepant lesions had significantly smaller multiparametric MRI-measured cancer volumes ( P < 0.05). Conclusion Knowledge of discrepant GS in MRI-US fusion biopsy is important, and a careful approach is needed to reduce this discrepancy.