SHG microscopy was successfully used to characterize the orientation of sutural lamellae within corneal samples by means of an epi-detection scheme. In particular, the attention was focused on the organization and orientation of corneal collagen lamellae in the first 30 μm of the stromal layer below Bowman’s membrane. In fact, it is thought that the irregular orientation of these specific lamellae, an in particular the existence of so-called sutural lamellae, affects the mechanical properties of the cornea and is responsible of the overall corneal stiffness. The methodology was tested by acquiring image stacks of the central portion of the cornea in ex vivo samples of healthy corneas, keratoconic corneas and keratoconic corneas treated with cross-linking. These samples represent a good benchmark for testing the methodology, considering that keratoconus is an ophthalmic disease in which the cornea loses its stiffness and acquires an abnormal conical shape because of a different organization of sutural lamellae. The samples were first imaged and characterized on the basis of forward/backward SHG ratio, finding a different ratio in keratoconus with respect to both the healthy cornea and cross-linked keratoconus. The inclination of corneal sutural lamellae was then characterized by means of a three-dimensional correlation analysis on SHG images, acquired using a backward detection geometry. Such method provided good discrimination capabilities, demonstrating that this approach can be used not only for diagnosing keratoconus in a very early stage, but also for performing treatment follow-up on cross-linked corneas.