Macrophages contribute to secondary damage and repair after central nervous system (CNS) injury. Micron-sized paramagnetic iron oxide (MPIO) particles can label macrophages in situ, facilitating three-dimensional (3D) mapping of macrophage accumulation following traumatic brain injury (TBI), via ex vivo magnetic resonance microscopy (MRM) and in vivo monitoring with magnetic resonance imaging (MRI). MPIO particles were injected intravenously (iv; 4.5 mg Fe/Kg) in male C57BL/6J mice (n = 21). A controlled cortical impact (CCI) was delivered to the left parietal cortex. Five protocols were used in naive and injured mice to assess feasibility, specificity, and optimal labeling time. In vivo imaging was carried out at 4.7 Tesla (T). Brains were then excised for 3D MRM at 11.7 T. Triple-label immunofluorescence (MPIO via Dragon Green, macrophages via F480, and nuclei via 4,6-diamidino-2-phenylindole [DAPI]) of brain sections confirmed MPIO particles within macrophages. MRM of naives showed an even distribution of a small number of MPIO-labeled macrophages in the brain. MRM at 48-72 h after CCI and MPIO injection revealed MPIO-labeled macrophages accumulated in the trauma region. When MPIO particles were injected 6 days before CCI, MRM 48 h after CCI also revealed labeled cells at the injury site. In vivo studies of macrophage accumulation by MRI suggest that this approach is feasible, but requires additional optimization. We conclude that MPIO labeling and ex vivo MRM mapping of macrophage accumulation for assessment of TBI is readily accomplished. This new technique could serve as an adjunct to conventional MR approaches by defining inflammatory mechanisms and therapeutic efficacy of anti-inflammatory agents in experimental TBI.