The aim of this study was to assess the involvement of deep gray matter, hippocampal subfields, and ventricular changes in patients with amyotrophic lateral sclerosis (ALS). A total of 112 ALS patients and 60 healthy subjects participated. High-resolution T1-weighted images were acquired using a 3T MRI scanner. Thirty-nine patients underwent a follow-up scan. Volumetric and shape analyses of subcortical structures were performed, measures were correlated with clinical parameters, and longitudinal changes were assessed. At baseline, reduced hippocampal volumes (left: p = 0.007; right: p = 0.011) and larger inferior lateral ventricles (left: p = 0.013; right: p = 0.041) were found in patients compared to healthy controls. Longitudinal analyses demonstrated a significant decrease in volume of the right cornu ammonis 2/3 and 4/dentate gyrus and left presubiculum (p = 0.002, p = 0.045, p < 0.001), and a significant increase in the ventricular volume in the lateral (left: p < 0.001; right: p < 0.001), 3rd (p < 0.001) and 4th (p = 0.001) ventricles. Larger ventricles were associated with a lower ALSFRS-R score (p = 0.021). In conclusion, ALS patients show signs of neurodegeneration of subcortical structures and ventricular enlargement. Subcortical involvement is progressive and correlates with clinical parameters, highlighting its role in the neurodegenerative process in ALS.