BACKGROUND AND PURPOSE: In the differential diagnostic workup of amyotrophic lateral sclerosis (ALS), magnetic resonance imaging (MRI) is primarily used to rule out significant differential diagnoses. So far, whole-brain T1-mapping has not been assessed as a diagnostic tool in this patient population. METHODS: We investigated the diagnostic potential of a novel T1-mapping method based on real-time MRI with 0.5 mm in-plane resolution and 4s acquisition time per slice. The study included patients aged 18 to 90 years who met the revised El Escorial criteria for at least possible ALS. T1-relaxation times were measured along the corticospinal tract in predefined regions of interest. RESULTS: Twenty-nine ALS-patients and 43 control group patients (CG) were included in the study. Median ALS Functional Rating Scale revised (ALSFRS-R) was 37 (IQR, 35-44) points and the mean duration from symptom onset to MRI was 21 ± 17 (SD) months. ALS patients showed significantly higher T1-relaxation times in all ROIs compared to CG with mean differences in the hand knob of 50 ms ( CONCLUSIONS: T1-relaxation times along the corticospinal tract are significantly elevated in ALS patients compared to CG and associated with lower ALSFRS-R. These results imply the analysis of T1-relaxation times as a promising diagnostic tool that can distinguish ALS patients from the control group. Ongoing longitudinal studies may provide deeper insights into disease progression and the effects of therapeutic interventions.