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Objective To understand the morphology and anatomical data of the temporal bone of tree shrews through micro-computed tomography (micro-CT) and microdissection. Methods Skull specimens from 10 tree shrews were scanned using micro-CT examination. The acquired images were used for three-dimensional reconstruction and measurement using the Mimics 10.01 software. Twenty tree shrews were subjected to microdissection and the data were measured. Results Micro-CT and three-dimensional reconstruction could clearly define the three-dimensional spatial position of the ear structure. Micro-CT and microdissection showed that the otic vesicles of the tree shrews were located on both sides of posterior-inferior skull bone. The location of the otic vesicles was superficial, and the bone was thin. All of the structures of the middle and inner ear of the tree shrews were well developed. The ossicular chain was differentiated into the malleus, incus and stapes. The location of the three semi-circular canals of the tree shrews was superficial and easy to dissect. In vivo, the three semi-circular canals were easy to localize and the surface bone was thin. The contour and structure of the cochlea and number of cochlear turns were similar to those in humans. Conclusion This study could provide anatomical data to allow tree shrews to be used as animal models for studying ear diseases.