In some cases with uncertain renal tumour lesions, it would be helpful to perform biopsies for the preoperative differential diagnosis. In our study, we evaluated the benefit of multi-colour interphase fluorescence in situ hybridization (M-FISH) on fine-needle core biopsies in uncertain renal masses. We prospectively performed three ultrasound-guided percutaneous biopsies in 25 patients with indeterminate renal masses preoperatively. Histopathology was performed on two remaining cores samples. M-FISH was performed on one core for chromosomes 1, 2, 6, 9, 7, 17, the loci 3p24pter, and 3p13p14. After interphase FISH evaluation, we classified tumours and compared the results with histopathological findings. 16 were classified as renal malignancies: 14 (56%) clear cell renal cell carcinomas (RCCs), 1 papillary RCCs (4%), and 1 "adenocarcinoma" (4%). Seven patients (28%) had a benign tumour, i.e. 6 (24%) were oncocytomas and 1 was classified as leiomyoma (4%). In two cases (8%), no renal neoplasms were found. In 19 out of 21 cases (90.5%), the preoperative diagnostic fine-needle biopsy matched the final histological findings. The combination of histopathological examination and M-FISH leads to a higher (95.5 vs. 90.5%) diagnostic fidelity as histology alone. Ultrasound-guided percutaneous renal tumour biopsy is an accurate and safety method for the histopathologic evaluation of uncertain renal masses. The M-FISH represents a new highly sensitive and specific method to confirm histopathological classification in less than 24 h which can be used in routine laboratory diagnosis.