The highly organized spatial arrangement of sensory hair cells in the organ of Corti is essential for inner ear function. Here, we report a new analytical pipeline, based on optical clearing of tissue, for the construction of a single-cell resolution map of the organ of Corti. A sorbitol-based optical clearing method enabled imaging of the entire cochlea at subcellular resolution. High-fidelity detection and analysis of all hair cell positions along the entire longitudinal axis of the organ of Corti were performed automatically by machine learning–based pattern recognition. Application of this method to samples from young, adult, and noise-exposed mice extracted essential information regarding cellular pathology, including longitudinal and radial spatial characteristics of cell loss, implying that multiple mechanisms underlie clustered cell loss. Our method of cellular mapping is effective for system-level phenotyping of the organ of Corti under both physiological and pathological conditions.