Background Fatigue is associated with poor functional outcomes and increased mortality following stroke. Survivors identify fatigue as one of their key unmet needs. Despite the growing body of research into post-stroke fatigue, the specific neural mechanisms remain largely unknown. Aim This observational study aimed to identify resting state brain activity markers of post-stroke fatigue. Method Sixty-three stroke survivors (22 women; age 30–89 years; mean 67.5 ± 13.4 years) from the Cognition And Neocortical Volume After Stroke study, a cohort study examining cognition, mood, and brain volume in stroke survivors following ischemic stroke, underwent brain imaging three months post-stroke, including a 7-minute resting state functional magnetic resonance imaging. We calculated the fractional amplitude of low-frequency fluctuations, which is measured at the whole-brain level and can detect altered spontaneous neural activity of specific regions. Results Forty-five participants reported experiencing post-stroke fatigue as measured by an item on the Patient Health Questionnaire-9. Fatigued compared to non-fatigued participants demonstrated significantly lower resting-state activity in the calcarine cortex ( p < 0.001, cluster-corrected p_FDR = 0.009, k = 63) and lingual gyrus ( p < 0.001, cluster-corrected p_FDR = 0.025, k = 42) and significantly higher activity in the medial prefrontal cortex ( p < 0.001, cluster-corrected p_FDR = 0.03, k = 45). Conclusions Post-stroke fatigue is associated with posterior hypoactivity and prefrontal hyperactivity reflecting dysfunction within large-scale brain systems such as fronto-striatal-thalamic and frontal-occipital networks. These systems in turn might reflect a relationship between post-stroke fatigue and abnormalities in executive and visual functioning. This whole-brain resting-state study provides new targets for further investigation of post-stroke fatigue beyond the lesion approach.