Abstract Background Pediatric renal tumors are often heterogeneous lesions with variable regions of distinct histopathology. Direct comparison between in vivo imaging and ex vivo histopathology might be useful for identification of discriminating imaging features. Objective This feasibility study explored the use of a patient-specific three-dimensional (3D)-printed cutting guide to ensure correct alignment (orientation and slice thickness) between magnetic resonance imaging (MRI) and histopathology. Materials and methods Before total nephrectomy, a patient-specific cutting guide based on each patient’s preoperative renal MRI was generated and 3-D printed, to enable consistent transverse orientation of the histological specimen slices with MRI slices. This was expected to result in macroscopic slices of 5 mm each. The feasibility of the technique was determined qualitatively, through questionnaires administered to involved experts, and quantitatively, based on structured measurements including overlap calculation using the dice similarity coefficient. Results The cutting guide was used in eight Wilms tumor patients receiving a total nephrectomy, after preoperative chemotherapy. The median age at diagnosis was 50 months (range: 4–100 months). The positioning and slicing of the specimens were rated overall as easy and the median macroscopic slice thickness of each specimen ranged from 5 to 6 mm. Tumor consistency strongly influenced the practical application of the cutting guide. Digital correlation of a total of 32 slices resulted in a median dice similarity coefficient of 0.912 (range: 0.530–0.960). Conclusion We report the feasibility of a patient-specific 3-D-printed MRI-based cutting guide for pediatric renal tumors, allowing improvement of the correlation of MRI and histopathology in future studies.