Introduction: More than a quarter of tumours are missed by MRI/US fusion-guided biopsy, the majority are due to software-based mis-registration. Transrectal approaches to biopsy are typically performed in the lateral decubitus position, conversely diagnostic MRI is performed with the patient lying supine. Any position-related difference in prostate location or gland deformation could potentially exacerbate mis-registration at subsequent biopsy. Materials and methods: 15 healthy male volunteers (mean age 35.9 years, range 27–53) were included in this prospective, institutional review board-approved study. Each volunteer had an MRI performed in the supine position, followed by the second in the lateral decubitus position (mimicking a typical biopsy position). MRI images were co-registered and analysed in order to assess prostate translocation and distortion. Results: Whole prostate translocation of ≥ 5 mm was observed in 20% of patients and ≥ 3 mm in 60% of patients. When dividing the prostate into prostatic sectors, the prostatic base demonstrated the largest positional difference. When plotting the translocation directions with relative volume difference, there was a moderate negative correlation trend in the latero-lateral direction. Only minimal distortion was observed, with similar distortion among all prostatic sectors. Conclusion: Positional change affects the prostate translocation, however the effect on prostate distortion appears to be negligible. Prostate translocation in latero-lateral direction can be minimised with larger bladder volumes. Thereby, prostate translocation needs to be considered alongside software misregistration error, however positional change should not affect software registration of MRI/US fusion-guided prostate biopsy.