The main objective of this study is to evaluate the rate of anisotropy in leather samples using a non-destructive method based on a free-space microwave transmission ellipsometry. The aim is to characterize the leather internal structure by determining the orientation of collagen fibers that ensure its mechanical properties. For this purpose, sixteen leather samples taken from different parts of a calfskin are analyzed. Leather anisotropy is obtained through the measurement of its birefringence and dichroism. But due to the small thickness of the samples, we only consider the influence of the dichroism which is here more significant. The experiment shows that sample anisotropy is proportional to the dichroism and can be classified into two groups according to their degree of anisotropy. Therefore, samples from the neck, legs, and flanks are of high anisotropy compared to those from the butt and abutment which are of low anisotropy. Results also show that from fibers orientation with respect to the animal backbone taken as the axis of reference, it is possible to locate the area where the sample was taken as well as its quality related to its anisotropy.