Objectives: In contrast to two-dimensional planar images, a measuring point is hardly repeatedly determined in a CBCT image when alveolar bone loss is assessed. Thus, the aim of the present study was to propose a six-site measuring method, which is closely related to anatomical structure, for the evaluation of alveolar bone loss in CBCT images. Methods: 150 measuring points in 11 molars and 14 premolars from 6 patients (2 males and 4 females) were included. CBCT images of the teeth were acquired prior to periodontal surgery. Four observers measured the distances between cemento-enamel junctions and the apical bases of the periodontal bone defect at the mesio-buccal, mid-buccal, disto-buccal, mesio-lingual/palatal, mid-lingual/palatal and disto-lingual/palatal sites in CBCT images. Direct measurements of the six sites were correspondingly obtained in the subsequent periodontal surgeries. Differences between the distances measured in the CBCT images and during the surgery were analysed. Interobserver and intraobserver variances were tested. Results: No statistically significant difference was found between the surgical and CBCT measurements (p 5 0.84). Diagnostic coincidence rates of four observers were 86.7%, 87.3%, 88.7% and 88.0%, respectively. The interobserver (p 5 0.95) and intraobserver (p 5 0.30) variances were not significant. Conclusions: The six-site measuring method validated in the present study may be a useful three-dimensional measuring method for the evaluation of periodontal disease.