Mimulus guttatus DC. (yellow monkey-flower; Phrymaceae) is an important model species for ecological and evolutionary studies, being locally adapted to a wide range of elevation, moisture and temperature gradients, soil types, and pollinator availabilities. In order to advance this species as a model for evolutionary genetic studies, we have developed virus-induced gene silencing (VIGS) using the tobacco rattle virus (TRV) to assay gene function. We demonstrate the effectiveness of Agrobacterium-mediated VIGS in two divergent populations of M. guttatus, Iron Mountain 767 (IM767) and Point Reyes (PR). Plants infected with a fragment of the carotenoid biosynthesis pathway gene PHYTOENE DESATURASE (PDS) cloned into the TRV2 vector exhibited endogenous PDS silencing and photobleached phenotypes. We further assayed for VIGS-induced floral phenotypes by silencing paralogous genes putatively affecting floral symmetry, CYCLOIDEA1 (CYC1) and CYCLOIDEA2 (CYC2). Simultaneous silencing of CYC1 and CYC2 resulted in organ number defects in the petal and stamen whorls; silencing of CYC1 affected petal margin growth; and silencing of CYC2 had no effect on flower development. Infection with TRV2 and TRV1 is significantly higher and more pervasive in the IM767 versus the PR population and is more efficient after vacuum infiltration. These results demonstrate the efficacy of VIGS for determining the function of developmental genes, including those involved in ecologically important reproductive traits.