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In the production of polyacrylonitrile-based carbon fibers (PAN-CFs), preoxidation is a key step to improving the thermal stability of the fibers and forming their structural prototype. However, structural evaluation of PAN fibers during preoxidation remains ambiguous, partly due to a lack of in situ studies. This contribution reports an in situ study of the preoxidation of PAN fibers using wide-angle X-ray scattering and small-angle X-ray scattering techniques on a synchrotron radiation beamline. The structural changes of the skeleton and pores in the fibers are revealed and correspond to five kinds of diffraction spots (peaks) and two kinds of scattering streaks, respectively. The preoxidation shows obvious stages, and the corresponding possible mechanism is analyzed.