Amyotrophic lateral sclerosis (ALS) is a progressive motor syndrome with clinical evidence of upper and lower motor neuron dysfunction. Mirror movements (MM) in ALS have been reported and attributed to a disturbed transcallosal inhibition (TI). Hence, occurrence of MM in ALS might be explained by involvement of transcallosal projecting fibre tracts into the degenerative process of the motor system. Twenty-six consecutive ALS patients were studied by clinical investigation of MM and by transcranial magnetic stimulation testing of TI using evaluation of the ipsilateral silent period. MM were observed in 39% of ALS patients. There was a significant correlation between the revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) and occurrence of MM (correlation coefficient -0.315; p = 0.044). In conclusion, all MM patients had pathological TI at least in one hemisphere, which indicates involvement of transcallosally projecting output neurons in ALS patients, which in turn may be an early feature of the disease process with the potential of a diagnostic biomarker.