Skin barrier function can be assessed non-invasively, including by transepidermal water loss (TEWL), skin hydration, and sebum level. The aim of this study was to evaluate day-to-day variation in measurements of TEWL, skin hydration, and sebum level at various anatomic sites and the relationship between these parameters in normal dogs. Measurements were repeated five times on two separate days in five clinically normal Beagle dogs at seven anatomic sites, i.e., the left and right pinnae, left and right axillae, left and right groin areas, and ventrum. Coefficient of variation was used to show the variation in measurements. Correlations between each of the measurements were analyzed to determine the contribution of skin hydration and sebum level to TEWL. There was no variation in the measurements obtained according to time or anatomic site (P>0.05). The coefficient of variation was highest for sebum level (209.0 ± 81.8%) followed in descending order by skin hydration (62.7 ± 34.5%) and TEWL (41.1 ± 6.9%). Of the seven anatomic sites sampled, the left and right pinnae showed the lowest variation in repeated measurements for TEWL (39.2%), skin hydration (29.6%), and sebum level (75.5%). There was no significant relationship between the results for each measurement (P>0.05). Because of its relatively low variation on repeated measurement, TEWL might be the most useful way of evaluating skin condition in dogs.