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A materials’ structure and its evolution due to friction play a crucial role in understanding wear and related processes. So far, structural changes caused by friction are mostly studied using ex situ destructive characterization techniques, such as microscopy of post-mortem the prepared specimen by polishing and etching techniques. In this paper, the structural changes of AISI 321 austenitic stainless steel (ASS) during frictional loading were observed by the nondestructive operando method based on synchrotron X-ray diffraction (XRD). Although the martensitic transformation in AISI 321 steel starts at ca. -187 °C, frictional loading induces γ -(ε, α′) transformation in this alloy at room or even higher temperatures. The ε-martensite formation is observed only for a relatively short time. Subsequently, a mechanically-mixed layer (MML), composed mainly of the α′ phase, forms at the sample’s surface. Using XRD peak profile analysis, we observed the accumulation of dislocations, their ordering, and/or stress field shielding before and after phase transformations. The steady-state conditions are reached after ca. 69 friction cycles manifested in reaching the threshold values of the size of the coherent scattering regions (CSRs) and dislocation density in γ and α′ phases. For a better understanding of structural evolution, the microstructure of the sample was studied by scanning electron microscopy (SEM) after the experiment. The structure of the MML, its delamination, the formation of vortices, and carbide crushing are discussed.