Objective: To compare four different three-dimensional horizontal planes and detect anatomical landmarks so as to provide theoretical reference for horizontal reference plane constructed by three-dimensional cephalometry. Methods: The subjects of this study were 32 facial symmetry patients (menton from mid-sagittal plane ≤2 mm). Cone-bead computed tomography (CBCT) was obtained before orthodontic treatment, and the data were imported into Dolphin imaging soft in DICOM format. The sagittal plane was passing through the Nasion, Sella and Dent. Four horizontal reference planes were constructed by three points of bilateral porion and bilateral orbitale. Plane 1: horizontal reference plane constructed by right porion and bilateral orbitale. Plane 2: horizontal reference plane constructed by left porion and bilateral orbitale. Plane 3: horizontal reference plane constructed by bilateral porion and right orbitale. Plane 4: horizontal reference plane constructed by bilateral porion and left orbitale. Pitch, yaw, roll for four planes were measured three dimensionally. All the samples were measured two times by one judge at an interval of two weeks. The two times measuring results were evaluated with Intraclass correlation coefficient (ICC) for verifying reliability. The multiple sets of repeated measurement analysis were used to compare the four different planes. Based on ages, the samples were divided into two groups (group 1: ages 13 to 17, group 2: over 18 years), the mean and standard deviation of landmark coordinates measured with Dent as the origin point, the circumference formula was applied to calculate the change of landmark position generated by head rotation. Results: No significant differences of pitch, yaw and roll among the four planes (P=0.196, 0.314, and 0.341). One degree of pitch rotation made changes of porion and orbitale approximate 0.5 mm, and 1.6 mm, respectively. One degree of yaw rotation made changes of porion and orbitale approximate 1.1 mm, and 1.5 mm, respectively. One degree of roll rotation made changes of porion and orbitale approximate 1.2 mm, and 0.7 mm, respectively. Conclusion: There was no significant difference among the four horizontal planes constructed by any three points of bilateral orbitales and bilateral porions. It has the highest concordance using bilateral orbitales and one porion to construct horizontal plane in this study, probably the best option in clinical practice. Different head rotation generated different distance changes of anatomical landmarks.