The use of a self-inflating tissue expander is a technique to stretch cutaneous tissues for potential use in reconstructive skin surgeries. This study investigates the mechanical properties of horse skin stretched by the subcutaneous implantation of anisotropic tissue expanders at the forehead, right shoulder, and dorsomedial part of the cannon region of the right forelimb in six (n = 6) horses. After 14 days of skin expansion, expanded and normal (control) skin samples were harvested and their mechanical properties of elastic modulus (EM), maximum force (MF), maximum stress (MSs) and maximum strain (MSr) were evaluated using uniaxial tension test. The expanded skin from shoulder area has higher EM, MSs, MSr and MF than the normal skin when compared to the forehead and lower forelimb. Statistically, there was a significant (P= .02) mean difference for MSs between the expanded shoulder and lower forelimb skin, but the pairwise comparison of EM, MSr and MF showed no significant difference between the locations. The overall effect of locations on EM and MSs was statistically significant (P < .05), however, there was no overall effect of horse factor, treatment factor (normal and expanded skin) and location interaction on the EM, MSS, MF and MSr. In conclusion, the expanded skin from the frontal head and the distal limb are less elastic (stiffer) compared to that of the expanded skin of the shoulder, thus anatomical location of the skin has some degree of effect on EM and MSs.