Skin is a multilayered multiscale composite material with a range of mechanical and biochemical functions. The mechanical properties of dermis are important to understand in order to improve and compare on-going in vitro experiments to physiological conditions, especially as the mechanical properties of the dermis can play a crucial role in determining cell behaviour. Spatial and isotropy variations in dermal mechanics are thus critical in such understanding of complex skin structures. Atomic force microscopy (AFM) based indentation was used in this study to quantify the three dimensional mechanical properties of skin at nanoscale resolution over micrometre length scales. A range of preparation methods was examined and a mechanically non-evasive freeze sectioning followed by thawing method was found to be suitable for the AFM studies. Subsequent mechanical evaluations established macroscale isotropy of the dermis with the ground substance of the dermis dominating the mechanical response. Mechanical analysis was extended to show significant variation in the elastic modulus of the dermis between anatomical locations that suggest changes in the physiological environment influence local mechanical properties. Our results highlight dependence between an isotropic mechanical response of the dermal microenvironment at the nanoscale and anatomical location that define the variable mechanical behaviour of the dermis.