The objective of this study was to determine central corneal thickness (CCT) and the intra- and interuser reliability using a portable spectral-domain optical coherence tomography (SD-OCT) device in canine eyes. Twenty clinically normal dogs were examined. CCT measurements were obtained from both eyes of each animal three times by two operators in succession. The CCT was automatically calculated using the pachymetry software generated from eight radial scans, 6 mm in length. Mean canine CCT was 606.83±39.45 μm for all eyes examined. There was no significant difference in CCT based on the eye examined (OD vs OS), age or gender of the animal. There was no significant difference in CCT between replicates performed by the same operator; however, a small but significant difference was noted in CCT between operators. The mean difference in CCT between operators was 1.9 μm (P=0.03). The coefficient of variation for each user and between users was very low (range 0.64-1.7 per cent). The intraclass correlation coefficient comparing operators was 0.975. Based on these results, the SD-OCT device evaluated is capable of obtaining precise CCT measurements with excellent intra- and interoperator reliability in canine eyes.