The manner in which X-rays are scattered or diffracted by the cornea provides us with valuable insights into the fine structure of the corneal stroma. This is because when X-rays pass through a cornea a diffraction pattern is formed due to scattering from regularly arranged collagen molecules and fibrils that comprise the bulk of the stromal matrix. Collagen provides the cornea with most of its strength, and its proper organisation is believed to be important for tissue transparency. Ever since 1978, when the first X-ray diffraction patterns were obtained from the cornea using radiation from a powerful synchrotron source, biophysicists have recorded and analysed a huge number of X-ray diffraction patterns from many different corneas. This article aims to explain the ideas that underpin our use of X-ray diffraction to investigate corneal ultrastructure, and show how the knowledge gained to date has far-reaching implications for tissue biomechanics, disease changes and transparency.