In this study, the influence of different sterilization methods on the thermo-gelation and structural properties of xyloglucan hydrogels was investigated. Xyloglucan samples were treated by either 70% ethanol, 70% isopropanol, γ-irradiation (10 kGy) at room temperature, γ-irradiation (10 kGy) in dry ice or autoclaving. These samples were tested for sterility by incubation with sterile Lysogeny Broth (LB) at room temperature, 30 °C and 37 °C for 30 days. According to the results obtained, xyloglucan hydrogels were only effectively sterilized by autoclaving or by γ-irradiation either at room temperature or in dry ice. These samples were analyzed by rheology measurements and dynamic and static light scattering analysis. Gamma-irradiation at room temperature markedly changed the polymer structure, preventing thermo-gelation. Only autoclaving and γ-irradiation in dry ice preserved the rheological properties of the polymer. The sol–gel transition as a function of the temperature was similar for these samples and the control sample.