The purpose of this study was to investigate the structure and organization of collagen during the healing of penetrating rabbit corneal wounds. Full-thickness wounds were produced in both corneas of six New Zealand White rabbits with a 2-mm trephine. After 5, 10, and 16 months, wounds were examined using transmission electron microscopy and wide-angle synchrotron X-ray diffraction, which allowed the quantification of the relative amount of collagen and fibril orientation in different parts of the cornea. The intensity of the diffraction patterns taken from the wound edge was much higher than patterns collected at the wound center, indicating a greater amount of collagen near the wound edge. This collagen was shown to have a preferred orientation, forming a circular pattern around the wound. As the wounds healed, the amount of preferentially aligned collagen decreased and lamella formation and ordered fibrillar arrangement increased as shown by electron microscopy. The orientation of collagen fibrils in healing penetrating corneal wounds is not random. Instead, fibrils tend to be circularly arranged within and without the wound, gradually becoming more normal over time. We speculate that collagen is artefactually displaced during trephination, resulting in an abnormal alignment of the fibrils surrounding the wound edge, and that this influences the wound healing process.