Microfocus X-ray diffraction has been used to analyze interfacial stress transfer in two-phase polymer systems consisting of a highly oriented polymer fiber in an isotropic resin matrix. Synchrotron radiation has been used to obtain high-quality X-ray diffraction patterns from single poly-(p-phenylene benzobisoxazole) (PBO) and poly(p-phenylene terephthalamide) (PPTA) fibers in these model two-phase systems. Two different specimen geometries were studied: a fully embedded fiber composite and a microdroplet specimen. A procedure is demonstrated whereby well-defined diffraction patterns can be obtained, using a 5 μm diameter X-ray beam, from individual 12 μm diameter polymer fibers in over 1 mm thickness of resin by subtracting the scattering of the resin matrix from that of the fiber and matrix to obtain a diffraction pattern of the fiber only. Shifts of meridional diffraction peaks of the fibers were determined as a function of stress and were converted into crystal strain. The fiber stress was then determined from calibrations obtained from previous X-ray diffraction studies of fiber deformation. The point-to-point variation of fiber stress was mapped along the fibers in the specimens, and a force-balance approach was used to determine the shear stress at the fiber-matrix interface. This present study was concerned only with an optically transparent polymer matrix, but the possible extension of the technique to opaque matrices and other multiphase polymer systems is discussed.