Abstract Alveolar macrophages (AMs) originate from fetal liver and self-renew under homeostasis. However, exposure to environmental agents is known to alter alveolar macrophage populations by promoting self-renewal and/or by inducing recruitment and differentiation of bone marrow-derived circulating monocytes. However, the transcriptomic profiling of fetal- versus bone marrow-derived AMs populations remains awaited. Therefore, we conducted transcriptomic analysis on two in vitro-differentiated (bone marrow-derived macrophages (BMDMs) and fetal liver-derived macrophages (FLDMs)) and three alveolar macrophage (BM-chimera, FL-chimera and naive C57BL6/J mice) populations. We hypothesized that, due to their fetal-liver origin, FLDMs, FL-chimera and naïve alveolar macrophages exhibit comparable gene expression patterns. Interestingly, the Principal Component Analysis revealed separation of these three macrophage populations, suggesting that lung microenvironment as well as precursor origin are critical in shaping AM gene expression profile. This finding was further supported by differential gene expression analysis. Custom gene sets relevant to selective macrophage functions revealed distinct expression patterns in in vitro, naïve, and chimeric AMs. This study is likely to provide critical insights on how lung microenvironment and precursor origin define gene expression and associated biological pathways.