Asphalt cement will not last for a long time as the world encounters a diminishment in the crude oil. For sustainable, flexible pavement development, new resources can provide a contribution to replace it partially or entirely. In this study, asphalt was partially and entirely replaced by guayule resin as a bioresource by-product, extracted during the guayule natural rubber production. Crumb rubber modifier (CRM) was used as an asphalt enhancer. The Superpave grading system was followed at

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... intermediate, and low temperatures to evaluate such innovative binder for rutting, fatigue, and thermal cracking, respectively, in addition to viscosity. Therefore, the original, short-term aging and long-term aging were simulated using tank, rolling thin film oven, and pressure aging vessel materials. Additionally, component analysis using Fourier-transform infrared spectroscopy was provided to link the rheological properties with the chemical changes. Outcomes showed a relatively much lower viscosity of guayule in the same high-temperature asphalt grade indicating savings in plant energy consumption and reduced environmental emissions. CRM enhanced guayule, but not as much as asphalt, proven by polymeric component migration through liquid binder. This enhancement was reflected in the rheological performance besides other factors. As-received guayule seems to have high oxygen content proven by strong absorption peak intensities of oxidative bonds (e.g., Carbonyl and sulfoxide). Such pre-oxidation was negatively reflected in the intermediate- and low-temperature performance of guayule and guayule-based binders. However, the investigated guayule had potential to compensate for asphalt replacement in the presence of CRM by 23–42% by weight of blend.