Abstract Mouse CD8α+ dendritic cells (DCs) in lymphoid organs and CD103+ CD11b− DCs in nonlymphoid tissues share phenotypic and functional similarities, as well as a unique shared developmental dependence on the transcription factor Batf3. Human DCs resembling mouse CD8α+ DCs in phenotype and function have been identified in human blood, spleen, and tonsil. However, it is not clear whether such cells are also present in human nonlymphoid organs, and their equivalence to mouse CD8α+ DC has recently been questioned. Furthermore, the identification of “CD8α+ DC-like” cells across different tissues and species remains problematic because of the lack of a unique marker that can be used to unambiguously define lineage members. Here we show that mouse CD8α+ DCs and CD103+ CD11b− DCs can be defined by shared high expression of DNGR-1 (CLEC9A). We further show that DNGR-1 uniquely marks a CD11b− human DC population present in both lymphoid and nonlymphoid tissues of humans and humanized mice. Finally, we demonstrate that knockdown of Batf3 selectively prevents the development of DNGR-1+ human DCs in vitro. Thus, high expression of DNGR-1 specifically and universally identifies a unique DC subset in mouse and humans. Evolutionarily conserved Batf3 dependence justifies classification of DNGR-1hi DCs as a distinct DC lineage.