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The absence of germinal centers (GCs) in cartilaginous fishes lies at odds with data showing that nurse sharks can produce robust antigen-specific responses and affinity mature their B cell repertoires. To investigate this apparent incongruity, we performed RNA sequencing on single nuclei, allowing us to characterize the cell types present in the nurse shark spleen, and RNAscope to provide in situ cellular resolution of key marker gene expression following immunization with R-phycoerythrin (PE). We tracked PE to the splenic follicles where it co-localizes with CXCR5^high centrocyte-like B cells and a population of putative T follicular helper (Tfh) cells, surrounded by a peripheral ring of Ki67⁺ AID⁺ CXCR4⁺ centroblast-like B cells. Further, we reveal selection of mutations in B cell clones dissected from these follicles. We propose that the B cell sites identified here represent the evolutionary foundation of GCs, dating …