The reaction-diffusion (RD) system is widely assumed to account for many complex, self- organized pigmentation patterns in natural organisms. However, the specific configurations of such RD networks and how RD systems interact with positional information (i.e., prepatterns) that may specify the initiation conditions for the RD operation remain largely unknown. Here, we introduced a three-substance RD system underlying the formation of repetitive pigment spots and stripes in Mimulus flowers. It consists of an R2R3-MYB activator (NEGAN), an R3-MYB inhibitor (RTO), and a coactivator represented by two paralogous bHLH proteins. Through fine- scale genetic analyses, transgenic experiments, and computer simulations, we identified the causal loci contributing to the evolutionary transition from sparsely dispersed spots to longitudinal stripes. Genetic changes at these loci modulate the prepatterns of the activator and coactivator expression and the promoter activities of the inhibitor and one of the coactivator paralogs. Our findings highlight the importance of prepatterns towards a realistic description of RD systems in natural organisms, and reveal the genetic mechanism generating pattern variation through modulation of the kinetics of the RD system and its prepatterns.