Diffraction patterns of silk from several spider species have been obtained by synchrotron radiation using a beam size ≥10 μm. Single fiber diffraction patterns were obtained for fiber diameters down to a few microns. Diffraction patterns recorded with a 10 μm wide X-ray beam displayed fiber texture. The presence of two fractions of different crystallinity was confirmed for a single Nephila clavipes fiber. The orientation distribution of the polymer chains of the crystalline fraction along the fiber axis was found to be about 23° full-width at half maximum (fwhm). The azimuthal spread of the short-range order fraction was about 86° fwhm.