Macrophages are a heterogeneous cell population involved in tissue homeostasis, inflammation, and various pathologies. Although the major tissue-resident macrophage populations have been extensively studied, interstitial macrophages (IMs) residing within the tissue parenchyma remain poorly defined. Here we studied IMs from murine lung, fat, heart, and dermis. We identified two independent IM subpopulations that are conserved across tissues: Lyve1loMHCIIhiCX3CR1hi (Lyve1loMHCIIhi) and Lyve1hiMHCIIloCX3CR1lo (Lyve1hiMHCIIlo) monocyte-derived IMs, with distinct gene expression profiles, phenotypes, functions, and localizations. Using a new mouse model of inducible macrophage depletion (Slco2b1 flox/DTR), we found that the absence of Lyve1hiMHCIIlo IMs exacerbated experimental lung fibrosis. Thus, we demonstrate that two independent populations of IMs coexist across tissues and exhibit conserved niche-dependent functional programming.