Our aim was to measure PDL space width using periapical radiography, CBCT and micro-CT. Dry human mandibular bones (n = 36) consisting of 59 molar and 36 premolar teeth were used. Four image sets were obtained as follows: (i) photostimulable phosphor plate (PSP), periapical radiography, (ii) CBCT 0.075 mm voxel size, (iii) CBCT 0.2 mm voxel size and (iv) micro-CT 0.06 mm voxel size. Micro-CT images were considered as the gold standard. Four observers measured the PDL space widths twice. Intra- and interclass correlation coefficients (ICC) were calculated. Spearman's rho correlation was assessed. PDL space widths ranged between 0.16 and 0.28 mm. There was good reliability within and between observers (P < 0.01). There was no significant relationship between micro-CT and periapical imaging. (P > 0.05). Best observer performance was obtained from CBCT 0.075 mm voxel size images followed by CBCT 0.2 mm voxel size images.