Recently, high-energy, short-pulsed femtosecond lasers have been used to generate signals in a process termed second harmonic generation (SHG) specifically from collagen that can be used to obtain high-resolution images of collagen without the need to fix, process, or section tissues. Using SHG imaging microscopy, we have studied the 3-dimensional collagen organization of the cornea. In normal human cornea, 3-dimensional reconstructed images revealed that the organization of collagen lamellae at anterior stroma is highly interwoven and contains prominent transverse “sutural” lamellae that insert into the anterior limiting layer (Bowman layer) and extend deep into the anterior stroma. By comparison, corneal buttons from patients with keratoconus lack sutural lamellae, suggesting that these macroscopic structures may play roles in establishing the structural integrity and biomechanical strength of the anterior cornea and in the pathogenesis of keratoconus. SHG imaging was also used to visualize photodisrupted stromal collagen after IntraLASIK surgery in an animal model and discontinuous collagen lamellae pattern after laser-assisted in situ keratomileusis surgery. Thus, SHG imaging of corneal stromal collagen organization has the potential to provide novel information about corneal physiology and pathobiology.