Diagnosis of post-stroke epilepsy is often challenging because of a low incidence of epileptiform abnormalities on electroencephalography (EEG). Hence, this study evaluated whether postictal subtraction single-photon emission computed tomography (SPECT) could visualize epileptic activity and act as a diagnostic modality in post-stroke epilepsy. Fifty post-stroke epilepsy patients, who had undergone Tc-99m-ECD SPECT twice (postictal and interictal), were enrolled. The postictal hyperperfusion area was identified by subtraction (postictal–interictal) SPECT and classified into two distribution types: superficial or deep-seated. Laterality and distribution of postictal hyperperfusion on subtraction SPECT were compared with stroke lesions, seizure symptoms, and epileptiform EEG findings. Forty-three of the 50 patients (86%) had hyperperfusion on subtraction SPECT and 26 (52%) had epileptiform EEG findings. Subtraction SPECT showed prolonged postictal hyperperfusion despite the relatively long interval between seizure end and postictal SPECT (median: 19.1 h, range: 2.2–112.5 h). The laterality of the hyperperfusion area had a high concordance rate with the laterality of stroke lesions (97.7%), seizure symptoms (91.9%), and epileptiform EEG findings (100%). Scalp EEG identified epileptiform activity more frequently in superficial type of SPECT, but less frequently in deep-seated type (both, P = 0.03). Postictal SPECT can be complementary to scalp EEG in endorsing the diagnosis and location of post-stroke epilepsy.