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This article presents the results of analyses of hair fibers from women with breast cancer using synchrotron-derived X-ray diffraction. These diffraction patterns contained a new feature superimposed on the normal diffraction pattern of α-keratin. The feature appeared as a ring with a molecular spacing determined to be 4.76 ± 0.07 nm. This feature was not present in the diffraction patterns of hair from women without breast cancer as assessed by other routine clinical diagnostic techniques. Furthermore, different hairs from the same subject analysed on two different synchrotron beamlines give remarkably consistent diffraction patterns. Previous studies by other investigators have suggested that analysis of X-ray diffraction patterns of hair can reveal the presence of breast cancer in clinical and preclinical trials. This finding, however, has not been independently confirmed. The methodologies of sample handling, sample exposure and image analysis are known to be vital. We discuss some of these issues and provide a detailed description of the methodology employed for the sample handling and image analysis and new methodologies developed from this work. We conclude that X-ray diffraction of hair has the potential to provide a non-invasive test for the presence of breast cancer.