Identification and classification of B cell subpopulations has been shown to be challenging and inconsistent among different species. Our study tested aspects of ontogeny, phenotype, tissue distribution, and function of equine CD5hi B cells, which represented a greater proportion of B cells early in development and in the peritoneal cavity. CD5hi and CD5lo B cells differentially expressed B cell markers (CD2, CD21, IgM) measured using flow cytometry, but similar mRNA expression of signature genes (DGKA, FGL2, PAX5, IGHM, IL10) measured using quantitative RT-PCR. Sequencing lambda light chain segments revealed that CD5hi B cells generated diverse immunoglobulin repertoires, and more frequently bound to fluorescence-labeled phosphorylcholine. This study shows developmental characteristics and tissue distribution of a newly described subpopulation of B cells in the horse.