Mast cells (MCs) are tissue-resident immune cells that exhibit homeostatic and neuron-associated functions. Here, we combined whole-tissue imaging and single-cell RNA sequencing datasets to generate a pan-organ analysis of MCs in mice and humans at steady state. In mice, we identify two mutually exclusive MC populations, MrgprB2+ connective tissue–type MCs and MrgprB2neg mucosal-type MCs, with specific transcriptomic core signatures. While MrgprB2+ MCs develop in utero independently of the bone marrow, MrgprB2neg MCs develop after birth and are renewed by bone marrow progenitors. In humans, we unbiasedly identify six MC clusters/states (MC1–6) distributed across 12 organs with different transcriptomic core signatures. MC1 are preferentially enriched in the skin and lungs, MC2, MC3, and MC4 in the skin and bladder, MC5 in the lymph node and vasculature, and MC6 in the trachea and lungs. This comprehensive analysis offers valuable insights into the natural diversity of MC subtypes in both mice and humans.