Purpose: To assess the difference between renal mass biopsy (RMB) performed either before or during the ablation procedure. Methods: A retrospective multicenter study was performed in patients with a cT1 renal mass treated with ablation between January 2007 and July 2019, including a search in the national pathology database for patients with a RMB planned for ablation. Patient and tumor characteristics and information on malignant, benign, and non-diagnostic biopsy results were collected to establish rates of overtreatment and number of ablations avoided in case of benign or non-diagnostic histology. Results: RMB was performed in 714 patients, of which 231 patients received biopsy before planned ablation, and 483 patients at the time of ablation. Pathology results before ablation were malignant in 63% (145/231), benign in 20% (46/231) and non-diagnostic in 17% (40/231). Pathology results at the time of ablation were malignant in 67.5% (326/483), benign in 16.8% (81/483) and non-diagnostic in 15.7% (76/483), leading to a total of 32.5% of ablation of benign or non-diagnostic lesions. Of the patients with a benign biopsy obtained before ablation, 80.4% (37/46) chose not to undergo ablation. Patients with inconclusive biopsy before planned ablation chose an informed individualized approach including ablation, repeated biopsy, or no intervention in 56%, 34% and 10%. Conclusion: This study emphasizes the importance of obtaining a biopsy prior to the ablation procedure in a separate session to lower the rate of potentially unnecessary ablations.