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Summary: A comprehensive cellular anatomy of normal human prostate is essential for solving the cellular origins of benign prostatic hyperplasia and prostate cancer. The tools used to analyze the contribution of individual cell types are not robust. We provide a cellular atlas of the young adult human prostate and prostatic urethra using an iterative process of single-cell RNA sequencing (scRNA-seq) and flow cytometry on ∼98,000 cells taken from different anatomical regions. Immunohistochemistry with newly derived cell type-specific markers revealed the distribution of each epithelial and stromal cell type on whole mounts, revising our understanding of zonal anatomy. Based on discovered cell surface markers, flow cytometry antibody panels were designed to improve the purification of each cell type, with each gate confirmed by scRNA-seq. The molecular classification, anatomical distribution, and purification tools for each cell type in the human prostate create a powerful resource for experimental design in human prostate disease. : Using single-cell RNA sequencing, immunofluorescence, and flow cytometry, Henry et al. create a cellular anatomy of the normal human prostate and provide the tools to identify, isolate, and localize every cell type. They identify two additional epithelial cell types enriched in the prostatic urethra and proximal prostatic ducts. Keywords: human prostate, zonal anatomy, single-cell RNA sequencing, flow cytometry, prostate cancer, benign prostatic hyperplasia, human cell atlas, prostate epithelia, prostate stroma, GUDMAP