Inhomogeneous Magnetization Transfer (ihMT) is a development from the MT MRI technique. IhMT can be considered as a dipolar order relaxation time (T 1D ) weighted imaging modality whose signal has shown an enhanced selectivity for myelin-rich structures. However, a formal validation of the ihMT sensitivity relative to a gold standard myelin density measurement has not yet been reported. To address this need, we compared ihMT MRI with green fluorescence protein (GFP) microscopy, in a study performed on genetically-modified plp-GFP mice, considered as a reference technique for myelin-content assessment. Various ihMT protocols consisting of variable T 1D -filtering and radiofrequency power temporal distributions, were used for comparison with fluorescence microscopy. Strong and significant linear relationships (r 2 (0.87–0.96), p < 0.0001) were found between GFP and ihMT ratio signals across brain regions for all tested protocol variants. Conventional MT ratios showed weaker correlations (r 2 (0.24–0.78), p ≤ 0.02) and a much larger signal fraction unrelated to myelin, hence corresponding to a much lower specificity for myelin. T 1D -filtering reduced the ihMT signal fraction not attributed to myelin by almost twofold relative to zero filtering suggesting that at least half of the unrelated signal has a substantially shorter T 1D than myelin. Overall, these results strongly support the sensitivity of ihMT to myelin content.