Dendritic cells are immune sentinels that initiate and regulate adaptive immune responses. Transitional dendritic cells (tDCs) represent a newly defined subset that exists on a transcriptional and phenotypic continuum between plasmacytoid dendritic cells (pDCs) and conventional DCs (cDCs). However, little is known about their localization in highly organized lymphoid organs such as the spleen. Using flow cytometry and immunofluorescence in genetically modified mouse models, we developed an imaging strategy to identify and distinguish splenic pDCs and tDCs. Splenic tDCs and pDCs, unlike cDC1s and cDC2s, share expression of the transcription factor TCF4 but differentially express surface markers CD11c and PDCA-1. We therefore defined tDCs as TCF4+PDCA-1-CD11c+ and pDCs as TCF4+PDCA-1+CD11c- for microscopy analysis. Both pDCs and tDCs are found in the splenic red pulp at steady state. In contrast, their distribution in other splenic compartments differs: tDCs preferentially occupy the marginal zone, whereas pDCs preferentially occupy the T-cell zone. We further validated pDC and tDC splenic localization using subset-specific depletion models. The marginal zone and the T-cell zone serve distinct immunological functions, with the former representing a major site of blood-borne antigen capture and the latter supporting T-cell priming. Thus, despite their developmental relationship, the distinct localization of pDCs and tDCs may position these populations to perform distinct functions during immune responses.