Previous studies failed to detect reduced value of the amplitude of low frequency fluctuation (ALFF) derived from resting state functional magnetic resonance imaging in the primary motor cortex in amyotrophic lateral sclerosis (ALS) though primary motor cortex was mainly affected with ALS. We aimed to investigate the cause of masking the abnormality in the primary motor cortex in ALS and usefulness of ALFF for differential diagnosis among diseases showing muscle weakness. We enrolled ten patients with ALS and eleven disease controls showing muscle weakness. Voxel-wise analysis revealed that significant reduction of ALFF value was present in the right sensorimotor cortex in ALS. There was a significant negative correlation between ALFF value in the right sensorimotor cortex and fractional anisotropy (FA) value in the posterior limbs of the internal capsule (PLIC). For a diagnostic tool, the area under receiver operating characteristic curve improved if the ALS patients with disease duration >1 year were excluded. The present findings raised the possibility of usefulness of ALFF value in the sensorimotor cortex for differential diagnosis of ALS, and supported the notion that adjustment for FA value in the PLIC could improve accuracy.