•Small-sized fasciculation potentials detected with EMG may be undetectable with ultrasonography.•Muscle strength between the muscles with and without fasciculations was not different.•Both EMG and ultrasonography should be used for diagnosis of amyotrophic lateral sclerosis. Fasciculation potentials (FP) are an important consideration in the electrophysiological diagnosis of ALS. Muscle ultrasonography (MUS) has a higher sensitivity in detecting fasciculations than electromyography (EMG), while in some cases, it is unable to detect EMG-detected fasciculations. We aimed to investigate the differences of FP between the muscles with and without MUS-detected fasciculations (MUS-fas). Thirty-one consecutive patients with sporadic ALS were prospectively recruited and in those, both needle EMG and MUS were performed. Analyses of the amplitude, duration, and number of phases of EMG-detected FPs were performed for seven muscles per patient, and results were compared between the muscles with and without MUS-fas in the total cohort. The mean amplitude and phase number of FP were significantly lower in patients with EMG-detected FP alone (0.39 ± 0.25 mV and 3.21 ± 0.88, respectively) than in those with both FP and MUS-fas (1.22 ± 0.92 mV and 3.74 ± 1.39, respectively; p < 0.0001 and p = 0.017, Welch’s t-test). Small FP may be undetectable with MUS. MUS cannot replace EMG in the diagnostic approach for ALS. Clinicians should use a combination of EMG and MUS for the detection and quantitative analysis of fasciculation in ALS.