Abstract Background and Aims Renal cortical interstitial fibrosis is a well-established prognostic indicator for renal health, conventionally estimated by biopsy. Our prior research has enhanced DW-MRI sequencing, facilitating precise distinction between the renal cortex and medulla. This has enabled the measurement of the cortico-medullary apparent diffusion coefficient difference (ΔADC), which inversely correlates with the extent of histologically assessed interstitial fibrosis. The present study is designed to validate these findings, assess their reproducibility in a separate institution, and ascertain their significance in delineated subgroups, particularly between allograft recipients and native kidney patients. Method From 2014 to 2023, we orchestrated a prospective cohort study encompassing 309 patients diagnosed with chronic kidney disease (n = 106) or those with a renal allograft (n = 203). Each patient had undergone a biopsy, subsequent to which a multimodal magnetic resonance imaging (MRI) was systematically conducted in the following weeks. Initially, participants were recruited from Geneva hospitals, with subsequent inclusion of patients from Lausanne in Switzerland. The study's objective was to evaluate the correlation between ΔADC and the degree of renal fibrosis at biopsy. We performed subgroup analyses (per center, native/allograft kidneys) and applied linear regression models to investigate these associations rigorously. Results We included 270 participants in Geneva and 39 patients in Lausanne. A significant negative correlation was observed between ΔADC and the degree of fibrosis across the entire cohort (R = −0.41, p < 0.001). Subgroup analyses yielded consistent findings in both Geneva (R = −0.43, p < 0.001) and Lausanne University Hospitals (R = −0.35, p < 0.001). Furthermore, this association remained significant within subgroups of both native and allograft kidneys (respectively: R = −0.48, p < 0.001 and R = −0.38, p < 0.001). Conclusion Our findings reinforce the link between ΔADC and renal fibrosis, indicating that this association is consistent regardless of the center or kidney type (native or allograft). This supports the potential utility of diffusion MRI as a non-invasive tool for the assessment of renal fibrosis.