Aim Renal fibrosis (RF) is a well‐known marker of chronic kidney disease (CKD) progression. However, renal biopsy is an available tool for evaluation of RF, non‐invasive tools are needed not only to detect but also to monitor the progression of fibrosis. The aim of this study is to evaluate the role of diffusion tensor imaging (DTI) in the assessment of renal dysfunction and RF in patients with renal disease. Methods Fifty‐six patients with renal disorders and 22 healthy controls were recruited. All participants underwent DTI. Renal biopsy was performed for all patients. Mean renal medullary and cortical fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values were compared between patients and healthy controls and correlated to serum creatinine (SCr), estimated glomerular filtration rate (eGFR), 24‐h urinary protein (24h‐UPRO) and renal histopathological scores. Results Cortical FA values were significantly higher (P = .001), while cortical ADC values were significantly lower in the patients’ group (P = .002). Cortical FA values positively correlated to SCr (P = .006) and negatively correlated to eGFR (P = .03), while cortical ADC negatively correlated to percentage of sclerotic glomeruli, atrophic tubules and interstitial fibrosis (P = .001 for all variables). Medullary ADC negatively correlated to tubular atrophy (P = .02). The diagnostic performance of DTI for detecting RF was supported by ROC curve. Multiple linear regression analysis revealed that the mean cortex ADC was significantly decreased by 0.199 mg/dL for patients with >50% glomerulosclerosis in renal biopsy. Conclusion DTI appears to represent a valuable tool for the non‐invasive assessment of renal dysfunction and renal fibrosis.