Some explants produce variably colored callus with different textures, which usually fail to regenerate complete plantlets. In addition, infection of leaf explants with Agrobacterium reduces callus induction and regeneration rates ( Dong et al., 2006). The present investigation was initiated to develop an efficient, fast and reproducible Agrobacterium-mediated transformation system of guayule using highly regenerating callus (HRC). The plant selection marker nptII gene was used to optimize the transformation. Agrobacterium-mediated transformation efficiency of HRC was analyzed with PCR. Transgene integration and translation was confirmed with Southern blot and western blot analysis respectively. The optimized protocol should hasten the routine transformation and improvement of guayule via metabolic engineering approaches for enhancing the latex yield ( Kumar et al., 2012).