Previous studies have revealed significant changes in electroencephalogram (EEG) microstates in neuropsychiatric diseases, including schizophrenia, depression, and dementia. To explore the resting-state EEG microstate with amputation, we collected the EEG datasets from 15 patients with lower limb amputation and 20 healthy controls. Then, we analyzed the parameters of four classical EEG microstates (A–D) between the two groups. Specifically, the parameters were statistically analyzed, including duration, occurrence rate, time coverage, and transition rate. According to the results, the duration of microstate C (t = 2.95, p = 0.005) in the lower limb amputation group was significantly smaller compared with the control group, while the occurrence rate of microstate B (t = –2.22, p = 0.03) and D (t = –3.35, p = 0.002) were significantly larger in the lower limb amputation group. In addition, the transition rate of microstate differed significantly in AC, CA, DB between the two groups. Our results implied: (1) amputation has changed the resting-state EEG microstate; (2) EEG microstate analysis can be an approach to explore the alteration of cortical function.