Background: Diffusion tensor imaging (DTI) has found clinical applications in the evaluation of the central nervous system and has been extensively used to image peripheral neuropathy. However, few studies have focused on lumbosacral nerve root fiber damage in diabetic peripheral neuropathy (DPN). The aim of the study was to evaluate whether DTI of the lumbosacral nerve roots can be used to detect DPN. Methods: Thirty-two type 2 diabetic patients with DPN and thirty healthy controls (HCs) were investigated with a 3T MRI scanner. DTI with tractography of the L4, L5, and S1 nerve roots was performed. Anatomical fusion with the axial T2 sequences was used to provide correlating anatomical information. Mean fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values were measured from tractography images and compared between groups. Diagnostic value was assessed using receiver operating characteristic (ROC) analysis. The Pearson correlation coefficient was used to explore the correlation between DTI parameters and clinical data and the nerve conduction study (NCS) in the DPN group. Results: In the DPN group, FA was decreased (p< 0.001) and ADC was increased (p< 0.001) compared with the values of the HC group. FA displayed the best diagnostic accuracy, with an area under the ROC curve of 0.716. ADC was positively correlated with HbA1c level (r = 0.379,p= 0.024) in the DPN group. Conclusions: DTI of lumbosacral nerve roots demonstrates appreciable diagnostic accuracy in patients with DPN.