This study investigates the structural transformation from the γ-phase into the α-phase in bio-based nylon 5,6 fibers during in situ uniaxial stretching, using X-ray diffraction (XRD) and density functional theory (DFT) calculations. Initially, nylon 5,6 films exhibited a well-defined γ-phase crystalline structure, and the as-spun fibers also retained a γ-phase-dominant structure with partial coexistence of α-phase components. Due to the lattice similarity between the γ- and α-phases, phase separation was challenging in the direction perpendicular to the fiber axis (ab-plane). However, the analysis of the (004) diffraction peaks along the fiber axis (c-axis) enabled the quantitative evaluation of each crystalline component. As the stretching progressed, the α(004) peak intensity gradually increased, indicating a continuous γ-to-α structural transition. Furthermore, DFT calculations revealed that the α-phase has lower energy than the γ-phase, supporting the thermodynamic favorability of the phase transition during elongation. These results provide a comprehensive understanding of the crystalline structure and transformation mechanism in environmentally friendly nylon fibers from both experimental and theoretical perspectives, and offer foundational insights for developing nylon materials with desirable properties through the precise control of crystal phase structures.