Renal artery stenosis (RAS) promotes fibrosis by excessive and irreversible collagen deposition which may lead to end stage renal failure. Currently, invasive tissue biopsy is the main tool to assess fibrosis. Magnetization transfer imaging (MTI) is a MR-based technique that is sensitive to the interaction of macromolecules (e.g. collagen) and free water. The characteristics of these interactions are notoriously organ and tissue dependent. This study tested the hypothesis that using MTI, renal fibrosis in RAS can be detected. MTI was applied in mice (n=7) with unilateral RAS induced by partial occlusion of the renal artery. In off-resonance MTI, to achieve highest sensitivity, offset frequency, RF pulse power and bandwidth were optimized towards enhancing the contrast between the fibrotic and non-fibrotic tissue. Moreover, magnetization exchange rates (kf and kr) and the fractional size of the restricted magnetization (F), as markers of tissue molecular-morphological change, were estimated using steady-state free precession, on-resonance MTI. The optimal contrast for visual differentiation was achieved at offset frequency, RF pulse power, and effective bandwidth of 6.6kHz, 10μT, and 300Hz, respectively. On-resonance MT demonstrated significantly higher F and kf in the stenotic vs. the contra-lateral kidney. Therefore, off-resonance MT can qualitatively differentiate the fibrotic from the non-fibrotic tissue. Furthermore, kf and F may serve as biomarkers for kidney morphological changes caused by RAS.