Imaging biomarkers that can detect pathological changes at an early stage of multiple sclerosis (MS) may allow earlier therapeutic intervention with an improved outcome. Using a mouse model of MS, termed as experimental autoimmune encephalomyelitis (EAE), we performed chemical exchange saturation transfer (CEST) MRI at a very early stage before symptom onset (6 days post-induction) for assessment of changes in tissues that appear “normal” with conventional MRI. The collected CEST Z-spectra signals (Ssat /S0 ) were analyzed using a histogram-guided method to determine the contributions from various offset frequencies. Histogram analysis showed that EAE mice exhibit a more heterogeneous distribution with lower peak heights in the hindbrain compared with nave mice at saturation offsets of 1 and 2 ppm. At these two offsets, both the mean Ssat /S0 and the mean MTRasym values in the cerebellum and brain stem are significantly different between EAE and nave mice (P < 0.05). Immunofluorescent staining validated the presence of neuroinflammation, with IBA1-positive cells detected throughout the hindbrain including the cerebellum and brain stem. Follow-up MRI at the symptom onset (score = 1.5-2.5, 13 days post-induction) confirmed gadolinium-enhanced periventricular lesions. CEST Z-spectra signals also changed by this time. The proposed three-level histogram-oriented analysis is simple to execute and robust for detecting subtle changes in Z-spectra signals, which does not require a priori knowledge of damage locations or contributing offset components. CEST MRI signals at 1 and 2 ppm were sensitive to the subtle pathological changes at an early stage in EAE mice, and have potential as novel imaging biomarkers complementary to functional and physiological MRI measures. 2019 John Wiley & Sons, Ltd.