<p>Mature citrus leaves are less susceptible to infections and exhibit a higher response to diseases than younger leaves. However, the recalcitrance of mature leaves to <i>Agrobacterium</i>-mediated genetic transformation has hampered functional genomics research. In this study, we developed an efficient methodology for <i>Agrobacterium</i>-mediated transient transformation of mature leaves of Mexican lime (<i>Citrus aurantifolia</i>). We evaluated the effect on transient gene overexpression in mature Mexican lime leaves of different <i>Agrobacterium</i> strains and densities, chemical compounds to induce T-DNA transfer, surfactant and phytohormone concentrations, and pretreatment with microwounding. These conditions were tested first on detached leaves and then <i>in-planta</i>. Mexican lime leaves were inoculated by infiltration using a needleless syringe with <i>Agrobacterium carrying</i> a binary vector with the <i>RUBY</i> reporter. This vector converts tyrosine to bright red betalain, which can be assayed quickly and inexpensively. Pre-treatment with microwounding, dithiothreitol (100&#xa0;µM), and Silwet (0.02%) increased the transformation efficiency of 5-week-old Mexican lime leaves. This resulted in leaf areas with 3- and fivefold higher betalain accumulation in detached and attached leaves, respectively. Significantly increased transient gene overexpression was confirmed by qRT-PCR analysis. Here we optimize a method of <i>Agrobacterium</i>-mediated transient genetic transformation of mature leaves of Mexican lime, reporting for the first time the in situ transient expression in mature leaves, which may contribute to the use of genetic engineering in studies to functionally characterize genes in mature leaves of this important species.</p>