To precisely identify mouse resident alveolar macrophages (AMs) and bone marrow (BM)–derived macrophages, we developed a technique to separately label AMs and BM-derived macrophages with a fluorescent lipophilic dye followed by FACS. We showed that this technique overcomes issues in cell identification related to dynamic shifts in cell surface markers that occurs during lung inflammation. We then used this approach to track macrophage subsets at different time points after intratracheal (i.t.) instillation of Escherichia coli LPS. By isolating BM-derived macrophages and AMs, we demonstrated that BM-derived macrophages were enriched in expression of genes in signal transduction and immune system activation pathways whereas resident AMs were enriched in cellular processes, such as lysosome/phagosome pathways, efferocytosis, and metabolic pathways related to fatty acids and peroxisomes. Taken together, these data indicate that more accurate identification of macrophage origin can result in improved understanding of differential phenotypes and functions between AMs and BM-derived macrophages in the lungs.