Background: Alzheimer’s disease (AD) is associated with atrophy in entorhinal cortex (ERC), the hippocampus, and its subfields Cornu Ammonis 1 (CA1) and subiculum, which predict conversion from Mild Cognitive Impairment (MCI) to clinical AD. The stratum radiatum, lacunosum and moleculare (SRLM) are also important gateways involving ERC and CA1, which are affected by early AD pathology. Objective: To assess whether the SRLM is affected in MCI and AD. Methods: In this proof-of-concept study, 27 controls, 13 subjects with AD and 22 with MCI underwent 3T MRI. T1 maps were used for whole-hippocampal volumetry, T2 maps were segmented for hippocampal subfield areas, entorhinal cortex and subiculum thickness, and evaluated for SRLM integrity. Results: Significant CA1 atrophy and subiculum thinning were found in both AD and MCI compared to similarly aged controls. However, SRLM integrity was only significantly reduced in AD but not in MCI compared to controls. There were no significant differences in other hippocampal subfields (CA2, CA3/Dentate Gyrus) or ERC thickness between the groups. Finally, CA1 and CA3/DG areas and SRLM clarity were correlated with clinical and cognitive measurements of disease severity. Conclusion: Although this study was cross sectional, it suggests a progression of specific subfield changes from MCI to established AD that is associated with the reduced integrity of SRLM, which may reflect more widespread hippocampal involvement as the disease progresses and the relative preservation of SRLM in MCI. These results provide new MRI biomarkers for disease staging and understanding of the neurobiology in AD.