Literature revealed the potential of using guayule resin for asphalt cement replacement from the binder’s perspective. However, monitoring guayule resin through binder-aggregate mixture could disclose its performance through field. In this study, designated binders were employed to investigate the applicability of such an innovative replacer through mixture, which were neat asphalt and guayule-based binders (neat guayule, asphalt-rubber-guayule, guayule-rubber binders). Consecutively, field-simulated lab mixtures were prepared to investigate the major distresses. Moisture damage, rutting, fatigue cracking, and thermal cracking resistances were investigated using the modified Lottman (TSR) test, rut test by asphalt pavement analyzer (APA), semi-circular bending (SCB) test, and disk-shaped compact tension (DCT) test, respectively. Additionally, the Hamburg wheel-tracking (HWT) test was employed to evaluate moisture susceptibility and rutting resistance. Outcomes revealed that the neat guayule was susceptible to moisture damage at a 7% air content (V_a) when the TSR test was employed. In contrast, all investigated mixtures yielded perfect performances against moisture susceptibility under the HWT test. Guayule-based mixtures perfectly resisted rutting, as analyzed by the rut test and HWT test. Generally, changing parameters (e.g., V_a, rubber addition, and partial asphalt replacement by guayule and rubber) enhanced the guayule-based mixture resistance to rutting and moisture damage resulting in acceptable performances. Guayule-based mixture had a high fracture toughness at low temperatures, hence fatigue fracture resistance at intermediate temperatures. Neat guayule mixture with or without rubber addition did not entirely resist thermal fracture. However, partial asphalt replacement by guayule and rubber resisted the thermal fracture to a great extent.