(1) Background: Skin barrier function resides mostly in the stratum corneum, which consists of a protein component, the corneocyte (bricks), which provides a scaffold for the second component, the extracellular matrix, consisting of multilayers of lipids (mortar). These two components closely interact and this could be the basis for the differences in the biophysical properties of the skin between anatomical regions. So, the aim of this study was to compare skin microstructural properties between body sites. (2) Methods: A comparative study was conducted that included healthy individuals without previous skin diseases. Skin barrier function parameters and microtopography parameters (smoothness, roughness, desquamation, wrinkles, surface, volume, contrast, variance, homogeneity, anisotropy, total cell count, flaking index, skin surface hardness, brightness, deformability and friction) were measured on the forearm, cheek and palm. (3) Results: 44 participants were included in this study, with a mean age of 38.8 ± 15.0 years. Significant differences were found between body sites for 14 of the 15 parameters evaluated. Smoothness was higher on the forearm than on the cheek and palm (240.02 Sems vs. 348.16 vs. 408.19 Sems, p < 0.05). Hardness was higher on the palm than on the forearm and cheek (13.22 AU vs. 9.44 AU vs. 7.94 AU, p < 0.05). Moreover, we observed that sociodemographic characteristics such as age, sex, tobacco and/or alcohol use, influenced the parameters evaluated. (4) Conclusions: The differences in skin barrier function and microtopography between anatomical regions reflects the different structure of skin in each body part and could help to understand the influence of the sociodemographic characteristics on theses parameters. This information could be useful for comparison with pathological skin characteristics and for targeting new treatments.