OBJECTIVE: Hertwig's epithelial root sheath (HERS) acts as a signaling center that regulates the size, shape, and number of tooth roots. Therefore, understanding the anatomical changes in HERS during development is crucial for investigating its effect on root formation. However, the three-dimensional morphology of HERS and its changes during tooth root development remain largely unknown due to the limitations of traditional histological techniques. METHODS: We developed an improved tissue clearing method for mouse embryonic and early postnatal mandibles, designated as a mini Transparent Embedding Solvent System (miniTESOS), based on the Transparent Embedding Solvent System (TESOS). We applied this method to the mouse line to systematically investigate the spatiotemporal dynamics of HERS at the cellular level during the development of the roots of mandibular first molars (MM1) and mandibular second molars (MM2). Additionally, using MM1 as a model, we quantitatively investigated the spatiotemporal changes in HERS in the root bifurcation region during the development of two-rooted teeth. RESULTS: In the early stages of root development, differences in growth rates and developmental patterns between MM1 and MM2 were observed in the mesiodistal and buccolingual directions, from the initiation to the fusion of buccal and lingual HERS. In the elongation stage of two-rooted teeth, continuous HERS was found exclusively at the leading edge of the root, gradually decreasing in length as the root extended. In the root bifurcation area, HERS undergoes four developmental stages: initiation, elongation, contact, and complete fragmentation, each characterized by specific morphological features. CONCLUSION: This study improves the understanding of the alterations in HERS during root development and summarizes its developmental pattern in the root bifurcation region. The miniTESOS tissue clearing method provides a new strategy to investigate tooth development.