Abstract Background Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are expressed by microglia and infiltrating macrophages following ischemic stroke. Whereas IL-1β is primarily neurotoxic in ischemic stroke, TNF-α may have neurotoxic and/or neuroprotective effects. We investigated whether IL-1β and TNF-α are synthesized by overlapping or segregated populations of cells after ischemic stroke in mice. Methods We used flow cytometry and immunohistochemistry to examine cellular co-expression of IL-1β and TNF-α at 6, 12 and 24 hours after permanent middle cerebral artery occlusion in mice, validating the results by the use of bone marrow chimeric mice. Results We found that IL-1β and TNF-α were expressed in largely segregated populations of CD11b⁺CD45^dim microglia and CD11b⁺CD45^high macrophages, with cells expressing both cytokines only rarely. The number of Gr1⁺ granulocytes producing IL-1β or TNF-α was very low, and we observed no IL-1β- or TNF-α-expressing T cells or astrocytes. Conclusion Taken together, the results show that IL-1β and TNF-α are produced by largely segregated populations of microglia and macrophages after ischemic stroke in mice. Our findings provide evidence of a functional diversity among different subsets of microglia and macrophages that is potentially relevant to future design of anti-inflammatory therapies in stroke.