Background: Magnetization transfer saturation (MTsat) imaging was developed to reduce T1 dependence and improve specificity to myelin, compared to the widely used MT ratio (MTR) approach, while maintaining a feasible scan time. As MTsat imaging is an emerging technique, the reproducibility of MTsat compared to MTR must be evaluated. Purpose: To assess the test–retest reproducibility of MTR and MTsat in the mouse brain at 9.4 T and calculate sample sizes potentially required to detect effect sizes ranging from 6% to 14%. Study Type: Prospective. Subjects: Twelve healthy C57Bl/6 mice. Field Strength/Sequence: 9.4 T; magnetization transfer imaging using FLASH-3D Gradient Echo; T2-weighted TurboRARE spin echo. Assessment: All mice were scanned at two timepoints (5 days apart). MTR and MTsat maps were analyzed using mean region-of-interest (ROIs: corpus callosum [CC], internal capsule [IC], hippocampus [HC], cortex [CX], and thalamus [TH]), and whole brain voxel-wise analysis. Statistical Tests: Bland–Altman plots were used to assess biases between test–retest measurements. Test–retest reproducibility was evaluated via between and within-subject coefficients of variation (bsCV and wsCV, respectively). Sample sizes required were calculated (significance level: 95%; power: 80%), given effect sizes ranging from 6% to 14%, using both between and within-subject approaches. Results were considered statistically significant at P ≤ 0.05. Results: Bland–Altman plots showed negligible biases between test–retest sessions (MTR: 0.0009; MTsat: 0). ROI-based and voxel-wise CVs revealed high reproducibility for both MTR (ROI-bsCV/wsCV: CC—4.5/2.8%; IC—6.1/5.2%; HC—5.7/4.6%; CX—5.1/2.3%; TH—7.4/4.9%) and MTsat (ROI-bsCV/wsCV: CC—6.3/4.8%; IC—7.3/5.1%; HC—9.5/6.4%; CX—6.7/6.5%; TH—7.2/5.3%). With a sample size of 6, changes on the order of 15% could be detected in MTR and MTsat, both between and within subjects, while smaller changes (6%–8%) required sample sizes of 10–15 for MTR, and 15–20 for MTsat. Data Conclusion: MTsat exhibited comparable reproducibility to MTR, while providing sensitivity to myelin with less T1 dependence than MTR. Evidence Level: 2. Technical Efficacy: Stage 1.