Objective To analyze and compare the characteristics and differences of corneal endothelial cells of rhesus monkey and tree shrew eyes. Methods Corneal endothelial cells of 6 healthy rhesus monkeys (12 eyes) and 20 healthy tree shrews (40 eyes) were measured using a non-contact SP3000P specular microscope. Eight parameters were determined and compared with relevant parameters of human eyes reported in the literature, including minimum cell area (S_min), maximum cell area (S_max), average cell area (S_avg), standard deviation of cell area (SD), coefficient of variability (CV), cell density (CD), hexagonality percentage (HG%) and central corneal thickness (CCT). Results The imaging and measurement of all parameters could be completed in a short time both in rhesus monkeys and tree shrews. The time spent in the two kinds of animals was not significantly different. The CCT was (449.2±12.8) μm and (262.4±24.6) μm, S_min was (120.4±26.3) S/μm² and (153.2±42.9) S/μm², S_max was (705.0±130.8) S/μm² and (468.7±109.3) S/μm², S_avg was (351.1±26.1) and (295.4±18.9) S/μm², S_SD was (113.1±27.4) and (75.9±27.3) S/μm², CV was (31.9±6.0) and (25.3±8.3), CD was (2874.2±203.8) p/cell·mm^-2 and (3399.3±224.7) p/cell·mm^-2, and the HG% was (58.6±9.1) and (94.0±9.7) in the rhesus monkeys andt tree shrews, respectively. The differences of all the above parameters between rhesus monkeys and tree shrews were statistically significant (P<0.05 for all). The cornea of tree shrews was significantly thinner than that of rhesus monkeys. The area and coefficient of variability of tree shrews were smaller to those of rhesus monkeys, while the cell density and hexagonality percentage were higher than those of rhesus monkeys. Compared with human eyes, the CCT, CV and HG% in rhesus monkeys were highly similar, while the CD was lower than that of human eyes. The CCT in tree shrew was only 60% of the rhesus monkey eyes and 50% of human eyes, while the CD and Savg were similar to that of human eyes in the 10-20 years old group. Conclusions The morphology and parameters of corneal endothelial cells in rhesus monkeys and tree shrews are significantly different. There are similarities and differences among the human, rhesus monkey and tree shrew corneal endothelial cells. Both rhesus monkeys and tree shrews are appropriate experimental animals feasible for researches on human corneal endothelial diseases.